strenghten yourself and other person

The object is “nothing” outside consciousness.

Hegel says that objects, as we experience them, are really just reflections of our own consciousness.

They have no independent reality apart from how we know them. In that sense, they are “null” or “nothing” on their own.

This “nothingness” is actually meaningful.

Even though objects are “null,” this nullity helps consciousness understand itself.

By recognizing that the object is just a reflection of itself, consciousness confirms its own nature. So the object’s nullity has a positive role: it’s a mirror for self-awareness.

Consciousness only exists in knowing.

For Hegel, consciousness is defined by knowing.

Something exists “for consciousness” only if consciousness knows it. That’s why the object’s reality is tied to our awareness of it.

Objects are really “self-alienation.”

When we encounter an object, it seems separate from us. But Hegel says this separateness is illusory.

The object is just consciousness seeing itself as “other,” so in relating to the object, consciousness is interacting with itself in disguise.

Consciousness overcomes this alienation.

Finally, consciousness realizes that the object is only a reflection of itself.

It can “take back” this alienation—seeing that what appeared to be external is actually part of itself.

In this way, consciousness becomes “at home” with the object and with itself.

COME BACK TO THIS PARA

Marx is saying that in Hegel’s philosophy, everything real — people, things, the world — is treated only as ideas in the mind. The result is that human life and alienation appear only as mental or spiritual problems (forms of consciousness), instead of real, material conditions that people actually live through.

Phenomenology is a philosophical approach focused on the study of subjective, conscious experience and how the world appears to us.

What’s the “negation”?

In Marx’s dialectical way of thinking (influenced by Hegel), a negation means the denial or destruction of something.

Under capitalism, human life is “negated.”

People are alienated from their work, from each other, and from themselves.

Human potential and creativity are suppressed — so capitalism is a negation of true human life.

1. What’s the “negation of negation”?

If capitalism is the first negation (it destroys real human freedom), then communism is the negation of that negation — it overcomes alienation and restores humanity.

So communism is not just tearing down capitalism — it’s affirming real human life again, by freeing people from alienation

speaking through the voice of god?

Should only 87116010 be kept for E-bikes in the EU?

The creation and research for my project will follow a similar list of components. 1) Locate and find the data: I have to select artworks that I believe are rich in narrative, to allow for an expressive animation. 2) Research and collect: Find as much information about the narrative as possible, and collect as much context as I can. The more the better, as it brings more content for the story. 3) Analyze the Data and Visuals: What parts are the best for animation? What would bring the narrative to life the most? 4) Communicating the Story in the Data: Using Krita and bringing the project to light.

accepting only/mostly women of higher nobility to "assign to their proper place"?

insinuating her as the higher order?

My project ties into the themes of digital humanities as well. Digital humanities is how we apply digital technologies to study culture, history and the arts. Because my project requires a lot of research with these 3 categories, my work with Krita will integrate with the concept of digital humanities with archeology.

I observe that the journal talks about how Lewis and Clark and the people who accompanied them had decided to set out early and passed the front of the island opposite to where they had camped last night, they then had breakfast on the mouth of a large creek on the south side. They had set their course to being north west and a short distance from where they were at the mouth of the creek there were paintings and carving in the limestone rock with white red and blue flint of good quality. In which the native Americans had taken a great deal of. I interpret this as a way for the author to make a detailed entry and record of their experiences and by giving specific land marks and other details like their course the author is able to make it easier for someone to retrace their steps if they were to. I can connect this to the tertiary source in how it talks about how the main objectives of this journey were to gain geographic and scientific information on the land and its plant and animal life. A second goal was also in the diplomatic and commercial interest of the United States who wanted to find a water route to the pacific in order to gain a stronger position in the fur trade. This is seen in the highlighted section in how the author notes how the land is by noting the creek and the landmark of the paintings on the rock. Context: Jefferson had wanted to expand the united states and compete with Canada in the fur trade. He had wanted to do so earlier than the expedition of Lewis and Clark and attempted so before the Louisiana purchase in 1803. However the expedition came to fruition following the purchase in which Lewis and Clark along with their group were tasked with exploring the vast recently purchased land.

This is nothing but the truth! No matter how I frame the artworks through Kirta, in some way, shape or form, my own personal biases and touch get employed onto the pieces. Although I can claim that I will not try to separate any form of truth from the narratives featured in my project, just my personal decision on what to animate and what not to, changes the narrative into a personalized one. I have to be careful with my decision-making and what I choose to bring to life.

I would say scrolling is the greatest invention in modern social media, especially for short videos. In the past, people usually chose the video they would like to watch based on the cover page and the title. It's somewhat related to consequentialism, as users have a purpose or expectations before they click into a video. However, the scrolling algorithm is giving users a kind of 'surprise'. The user will never know what video will be the next one, so they will continue to scroll down to find something new and satisfy the sense of opening a mystery box. The scrolling algorithm kept users on their social media for a significant time, but the feature of short videos is lowering the quality of information that the user will receive.

Reading [e33], the inventor apologizes for infinite scroll because it removes stopping points and lengthens use. I propose an ethical design rule: build in “natural stops”—page ends, session timers, and a one-tap “take a break” card—enabled by default and easy to keep on. Platforms should have to justify any removal of these stops.

Reading the Wikipedia entry on AIM (AOL Instant Messenger) brought back a lot of nostalgia. AIM was my first experience with real-time online communication, and it shaped how my generation learned to talk online. The concept of having a screen name, an "away message," and even custom sounds for incoming messages was revolutionary at the time. What really stood out from the source was how AIM not only popularized online chatting, but also introduced early forms of status updates and even emoticons—features we now take for granted on modern platforms like WhatsApp, Discord, or Slack. It’s fascinating to see how many of today’s messaging habits were born out of tools like AIM. It makes me appreciate how even "outdated" tech plays a role in shaping current digital culture.

I cannot assess the impact of conspiracy theories on political movements, but considering the social context at the time, the prevalence of such theories created intense friction between the two parties, to the extent that I believe the public lost trust in the government. On the other hand, conspiracy theories also, to some degree, fueled popular movements and drove certain reforms.

The article discusses how conspiracy theories were common in 19th-century American politics, often used by parties to gain power and spread fear. It shows that political distrust and rumors have long been part of U.S. history.

This article shows the history of how black people were looking for a platform to express themselves, communicating with other black people, and spreading their culture to the society. They tried Facebook and some other platforms and finally realized that twitter is a platform that had more freedom and fun. From this article, I felt the power of social media. It brings people together and pushes the development of society and culture.

This article explores the history of the subcommunity that has emerged from the social media platform, Twitter. "Black Twitter" has created a space where Black individuals can share their thoughts and experiences with a community of people from similar backgrounds.

This article introduces the definition of scrolling and different types of scrolling. I was not trying to pay attention to the scrolling when I was viewing the website or watching a TV show. After I read this article, I found out how useful the scrolling was.

I actually found this article really interesting, since it spoke to things I feel myself and many other users have experienced online before. Many of us have rage clicked at old websites that refuse to load, even though there's no logical indication that brute force will somehow force the program to work. And cognitive or emotional friction is a very real issue, as sometimes when the website or UI is frustrating enough it's easier to just abandon it altogether.

This movie clip gives insight into Web 2.0. It talks about how the age of privacy is long gone and we as a society are not getting it back, foreshadowing how now everything on social media is for the public view. It talks about the early stages of Facebook and introducing online communication. Furthermore, they talk about discouraging introducing ads to Facebook, showing how Web 2.0 was about expanding as quickly as possible instead of immediate revenue.

This video humorously recounts the rivalry and conflicts between John Adams and Thomas Jefferson in American history. Though presented as comedy, it helped me grasp how their differing political philosophies shaped the nation's early development. This approach to teaching history through entertainment also highlights the diversity of social media content—information doesn't always need to be delivered in a formal, serious manner. Sometimes, a lighthearted approach can make people more willing to explore and discuss history.

To summarize this article, user friction is anything that prevents someone from using the website or app in the way that it is intended and how they want. Emotional friction occurs when the user has a complex negative emotional reaction caused by the site and is hindered from completing the task. Interaction friction happens when the site is not easy to navigate. Cognitive friction is when the site isn't setup in the way that a user is used to, and so they give up on trying to figure out the new terms.

I thought it was interesting how conspiracy theories were spread around for so long. It is a kind of political propaganda that somehow spreads faster when it is a little too crazy to believe. I feel like the creation of forum in the digital age aided the spread of conspiracy theories because people can easily find other people who believe the same conspiracy and they can spur each other on.

Social media like bilibili were using this strategy (bullet screen) to gather more and more users. Or, in other words, it was a special feature that allowed bilibili to stand out from other video-based social media platforms. The bullet screen is forming a culture and attracting users; however, the way of informal connections may cause some problems. For example, people could easily argue about a specific or minor issue because almost no punishment will be imposed, and people from the two sides will never meet each other offline. Sometimes it's ridiculous that two people who don't even know each other will become enemies online.

I support adding "friendly friction" to the UI. The pop-up before retweeting makes me hesitant. I also suggest offering adjustable friction levels (read timers, cooldowns for late-night posts). This would reduce impulsive spreading while still maintaining freedom of choice. Do you agree?

I think it is based on the users' intention. If they want more privacy, they could just provide their name. But for working apps like Linkin or dating apps, it would be more appropriate to provide more information such as age, school degree or working status.

Two ethics frameworks that would apply to the different ways social medias allow connections to others are Aztec Virtue Ethics and Relational Ethics. Each of these frameworks emphasize the importance of community and relationships to excel as an individual. The isolating nature of many social media sites directly opposes these philosophies. They would likely be against the individualized use of social media and advocate for not using social media or to use it in ways that help build or find community.

The Greeks mostly saw Africa as opposites to them, they saw the climate was vastly differant, they saw that the social constructs were different and also viewed them as barbaric.

Quality Management Plan

The Quality Management Plan defines how quality will be managed throughout the project. It ensures deliverables meet requirements and stakeholder expectations.

It includes:

• Quality standards & practices – which standards apply (e.g., ISO, company standards).
• Roles and responsibilities – who handles quality tasks and when.
• Processes and procedures – how quality will be checked, controlled, and improved.
• Decision reviews – confirming earlier quality-related choices are still valid.
• Quality meetings and reports – what will be discussed and documented.
• Quality metrics – how quality performance will be measured.
• Measurement points – which deliverables will be evaluated and when.

Quality Metrics

Quality metrics are specific, measurable indicators used to evaluate how well the project is performing against its quality goals. They are defined in advance so performance can be tracked objectively.

Examples:

• Number of change requests → shows how well the project was planned.
• Resource utilization variance → checks if resources are being used efficiently.
• Number of items that fail inspection → measures product or process quality.
• Number of software bugs → tracks defect rate for development projects.

✅ In short: The Quality Management Plan explains how quality will be ensured. Quality Metrics specify what will be measured to confirm that quality.

Reserve Analysis

A technique used to identify and include extra time or money (reserves) in the project budget or schedule to handle risks and uncertainties.

Types of Reserves:

1. Contingency Reserve (for Known Unknowns)

• Covers identified risks that might happen (e.g., delays, rework).
• Usually a percentage of the project budget or estimated using quantitative risk analysis.
• Controlled by: the project manager.
• Example: Adding 10% extra budget for possible material price increases.

2. Management Reserve (for Unknown Unknowns)

• Covers unforeseen risks — things you didn’t or couldn’t plan for.
• Not part of the cost baseline (released only with approval).
• Controlled by: senior management or sponsor.
• Example: Unexpected regulation change requiring redesign.

Cost of Quality (COQ)

Refers to the total cost of ensuring quality in a project. It includes:

• Cost of conformance – investment to prevent defects (e.g., training, inspections, testing).
• Cost of non-conformance – cost after defects occur (e.g., rework, warranty claims, loss of reputation).

✅ In short:

Reserve Analysis adds planned buffers for uncertainty — Contingency = known risks, Management = unknown risks, and COQ balances prevention costs vs. failure costs to ensure quality.

I agree with this because there is many tourists blogging about Africa when they don't know anything about their culture and are simply sharing their biased point of view.

These stay consistent with videos I see online that are people asking for help and tourists going around filming people not translating their culture.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

We thank the referees for taking time to review our manuscript. These reviews are positive, highlighting the novelty of our findings. The majority of comments are cosmetic, and we have added data in response to some technical points. We feel that some of the additional experiments proposed would not add significant methodological depth, and cross-commenting suggests that our referees agree. At present we are attempting antibody staining to quantify Tk peptide retention in the midgut, as per suggestion by reviewer #2.

We enclose our point-by-point response to each referee's points, below.

__Reviewer #1 __

• Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done?

• We have incorporated the requested information into legends for lifespan experiments.

• Do the interventions shorten lifespan relative to the axenic cohort? Or do they prevent lifespan extension by axenic conditions? Both statements are valid, and the authors need to be consistent in which one they use to avoid confusing the reader.

• We read these statements differently. The only experiment in which a genetic intervention prevented lifespan extension by axenic conditions is neuronal TkR86C knockdown (Figure 6B-C). Otherwise, microbiota shortened lifespan relative to axenic conditions, and genetic knockdowns extend blocked this effect (e.g. see lines 131-133). We have ensured that the framing is consistent throughout, with text edited at lines 198-199, 298-299, 311-312, 345-347, 408-409, 424-425, 450, 497-503.

• TkRNAi consistently reduces lipid levels in axenic flies (Figs 2E, 3D), essentially phenocopying the loss of lipid stores seen in control conventionally reared (CR) flies relative to control axenic. This suggests that the previously reported role of Tk in lipid storage - demonstrated through increased lipid levels in TkRNAi flies (Song et al (2014) Cell Rep 9(1): 40) - is dependent on the microbiota. In the absence of the microbiota TkRNAi reduces lipid levels. The lack of acknowledgement of this in the text is confusing

• We have added text at lines 219-222 to address this point. We agree that this effect is hard to interpret biologically, since expressing RNAi in axenics has no additional effect on Tk expression (Figure S7). Consequently we can only interpret this unexpected effect as a possible off-target effect of RU feeding on TAG, specific to axenic flies. However, this possibility does not void our conclusion, because an off-target dimunition of TAG cannot explain why CR flies accumulate TAG following TkRNAi We hope that our added text clarifies.

• *I have struggled to follow the authors logic in ablating the IPCs and feel a clear statement on what they expected the outcome to be would help the reader. *

• We have added the requested statement at lines 423-424, explaining that we expected the IPC ablation to render flies constitutively long-lived and non-responsive to A pomorum.

• *Can the authors clarify their logic in concluding a role for insulin signalling, and qualify this conclusion with appropriate consideration of alternative hypotheses? *

• We have added our logic at lines 449-454. In brief, we conclude involvement for insulin signalling because FoxO mutant lifespan does not respond to TkRNAi, and diminishes the lifespan-shortening effect of * pomorum*. However, we cannot state that the effects are direct because we do not have data that mechanistically connects Tk/TkR99D signalling directly in insulin-producing cells. The current evidence is most consistent with insulin signalling priming responses to microbiota/Tk/TkR99D, as per the newly-added text.

• Typographical errors

• We have remedied the highlighted errors, at lines 128-140.

• I'd encourage the authors to provide lifespan plots that enable comparison between all conditions

• We have plotted our figures in faceted boxes, because the number of survival curves that would need to be presented on the same axis (e.g. 16 for Figure 5) would not be intellegible. However we have ensured that axes on faceted plots are equivalent and with grid lines for comparison. Moreover, our approach using statistical coefficients (EMMs) enables direct quantitative comparison of the differences among conditions.

Reviewer #2

• Not…essential for publication…is it possible to look at Tk protein levels?

• We have acquired a small amount of anti-TK antibody and we will attempt to immunostain guts associated with * pomorum and L. brevis*. We are also attempting the equivalent experiment in mouse colon reared with/without a defined microbiota. These experiments are ongoing, but we note that the referee feels that the manuscript is a publishable unit whether these stainings succeed or not.

• it would be good to show that the bacterial levels are not impacted [by TkRNAi]

• We have quantified CFUs in CR flies upon ubiquitous TkRNAi (Figure S5), finding that the RNAi does not affect bacterial load. New text at lines 138-139 articulates this point.

• The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this?

• As per response to Reviewer #1, we have added text at lines 219-222 to address this point.

• Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned?

• We have added another experiment showing longevity upon knockdown in conventional flies, using an independent TkRNAi line (Figure S3).

• Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this

• This comment relates to Figure 7G. We do see an effect of the knockdown in this experiment, so we believe that the knockdown is effective. However the direction of response is not consistent with our hypothesis so the experiment is not informative about the role of these cells. We therefore feel there is little to be gained by testing efficacy of knockdown, which would also be technically challenging because the cells are a small population in a larger tissue which expresses the same transcripts elsewhere (i.e. necessitating FISH).

• Would it be possible to use antibodies for acetylated histones?

• The comment relates to Figure 4C-E. The proposed studies would be a significant amount of work because, to our knowledge, the specific histone marks which drive activation in TK+ cells remain unknown. On the other hand, we do not see how this information would enrich the present story, rather such experiments would appear to be the beginning of something new. We therefore agree with Reviewer #1 (in cross-commenting) that this additional work is not justified.

Reviewer #3

• *In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary. *

• We thank the reviewer sincerely for their keen eye, which has highlighted an error in the previous version of the figure. In revisiting this figure we have noticed, to our dismay, that the figures for GFP quantification were actually re-plots of the figures for (ac)K quantification. This error led to the discrepancy between statistics and graphics, which thankfully the reviewer noticed. We have revised the figure to remedy our error, and the statistics now match the boxplots and results text.

• Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association

• We selected Adh on the basis of our RNAseq analysis, which showed it was not different between AX and CV guts, whereas many commonly-used “housekeeping” genes were. We have now added a plot to demonstrate (Figure S2).

• The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference

• We have added the requested reference (Hung et al, 2020) at line 86.

• Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.

• We agree with reviewers 1-2 (in cross-commenting) that this proposal is non-trivial and not justified by the additional insight that would be gained. As described above, we are attempting to immunostain Tk, which if successful will provide a third line of evidence for regulation of Tk+ cells. However we note that we already have the strongest possible evidence for a role of these cells via genetic analysis (Figure 5).

• While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (ie. Tk transcription or EE activity upregulation) by the microbiome on males.

• As the reviewer recognises, maintaining axenic experiments for months on end is not trivial. Given the tendency for males either to simply mirror female responses to lifespan-extending interventions, or to not respond at all, we made the decision in our work to only study females. We have instead emphasised in the manuscript that results are from female flies.

• TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D.

• We disagree with this interpretation: the results do not show that TkR86C-RNAi recapitulates the effect of enteric Tk-RNAi. A potentially interesting interaction is apparent, but the data do not support a causal role for TkR86C. A causal role is supported only for TkR99D, knockdown of which recapitulates the longevity of axenic flies and TkRNAi flies. Therefore we feel that our current title is therefore justified by the data, and a more generic version would misrepresent our findings.

• The difference between "aging" and "lifespan" should also be addressed.

• The smurf phenotype is a well-established metric of healthspan. Moreover, lifespan is the leading aggregate measure of ageing. We therefore feel that the use of “ageing” in the title is appropriate.

• If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.

• Foxo nuclear localisation has already been shown in axenic flies (Shin et al, 2011). We have added text and citation at lines 402-403.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary

Marcu et al. demonstrate a gut-neuron axis that is required for the lifespan-shortening effects mediated by gut bacteria. They show that the presence of commensal bacteria-particularly Acetobacter pomorum-promotes Tk expression in the gut, which then binds to neuronal tachykinin receptors to shorten lifespan. Tk has also recently been reported to extend lifespan via adipokinetic hormone (Akh) signaling (Ahrentløv et al., Nat Metab 7, 2025), but the mechanism here appears distinct. The lifespan shortening by Ap via Tk seems to be partially dependent on foxo and independent of both insulin signaling and Akh-mediated lipid mobilization. Although the detailed mechanistic link to lifespan is not fully resolved, the experiment and its results clearly shows the involvement of the molecules tested. This work adds a valuable dimension to our growing understanding of how gut bacteria influence host longevity. However, there are some points that should be addressed.

1. Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.
2. In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary.
3. If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.
4. Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association and/or (2) compare the results using different genes as internal standard.
5. While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (ie. Tk transcription or EE activity upregulation) by the microbiome on males.
6. We also had some concerns regarding the wording of the title. Fig6B and C suggests that TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D. The difference between "aging" and "lifespan" should also be addressed. While the study shows a role for Tk in lifespan, assessment of aging phenotypes (eg. Climbing assay, ISC proliferation) beyond the smurf assay is required to make conclusions about aging.
7. The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference, if available.

Referees cross-commenting

I agree with the other reviewers that the study has been done very well and hence additional experiments are not mandatory to be published such as calcium imaging. However, I still believe that testing Tk's elevation by the Ap in males should greatly increase the generality of the finding, no matter what the outcome would be. Too many studies use only females.

Significance

General assessment

The main strength of this study is the careful and extensive lifespan analyses, which convincingly demonstrate the role of gut microbiota in regulating longevity. The authors clarify an important aspect of how microbial factors contribute to lifespan control. The main limitation is that the study primarily confirms the involvement of previously reported signaling pathways, without identifying novel molecular players or previously unrecognized mechanisms of lifespan regulation.

Advance

The lifespan-shortening effect of Acetobacter pomorum (Ap) has been reported previously, as has the lifespan-shortening effect of Tachykinin (Tk). However, this study is the first to link these two factors mechanistically, which represents a significant and original contribution to the field. The advance is primarily mechanistic, providing new insight into how microbial cues converge on host signaling pathways to influence ageing.

Audience

This work will be of particular interest to a specialized audience of basic researchers in ageing biology. It will also attract interest from microbiome researchers who are investigating host-microbe interactions and their physiological consequences. The findings will be useful as a conceptual framework for future mechanistic studies in this area.

Field of expertise

Drosophila ageing, lifespan, microbiome, metabolism

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

The main finding of this work is that microbiota impacts lifespan though regulating the expression of a gut hormone (Tk) which in turn acts on its receptor expressed on neurons. This conclusion is robust and based on a number of experimental observation, carefully using techniques in fly genetics and physiology: 1) microbiota regulates Tk expression, 2) lifespan reduction by microbiota is absent when Tk is knocked down in gut (specifically in the EEs), 3) Tk knockdown extends lifespan and this is recapitulated by knockdown of a Tk receptor in neurons. These key conclusions are very convincing. Additional data are presented detailing the relationship between Tk and insulin/IGF signalling and Akh in this context. These are two other important endocrine signalling pathways in flies. The presentation and analysis of the data are excellent.

There are only a few experiments or edits that I would suggest as important to confirm or refine the conclusions of this manuscript. These are:

1. When comparing the effects of microbiota (or single bacterial species) in different genetic backgrounds or experimental conditions, I think it would be good to show that the bacterial levels are not impacted by the other intervention(s). For example, the lifespan results observed in Figure 2A are consistent with Tk acting downstream of the microbes but also with Tk RNAi having an impact on the microbiota itself. I think this simple, additional control could be done for a few key experiments. Similarly, the authors could compare the two bacterial species to see if the differences in their effects come from different ability to colonise the flies.
2. The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this? Better clarification is required.
3. With respect to insulin signalling, all the experiments bar one indicate that insulin is mediating the effects of Tk. The one experiment that does not is using dilpGS to knock down TkR99D. Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this, but as a minimum I would be a bit more cautious with the interpretation of these data.
4. Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned? This would further clarify that there are no off-target effects that can account for the phenotypes.

There are a few other experiments that I could suggest as I think they could enrich the current manuscript, but I do not believe they are essential for publication: 5. The manuscript could be extended with a little more biochemical/cell biology analysis. For example, is it possible to look at Tk protein levels, Tk levels in circulation, or even TkR receptor activation or activation of its downstream signalling pathways? Comparing Ax and CR or Ap and CR one would expect to find differences consistent with the model proposed. This would add depth to the genetic analysis already conducted. Similarly, for insulin signalling - would it be possible to use some readout of the pathway activity and compare between Ax and CR or Ap and CR? 6. The authors use a pan-acetyl-K antibody but are specifically interested in acetylated histones. Would it be possible to use antibodies for acetylated histones? This would have the added benefit that one can confirm the changes are not in the levels of histones themselves. 7. I think the presentation of the results could be tightened a bit, with fewer sections and one figure per section.

Referees cross-commenting

Reviewer 1

I generally agree with this reviewer but for

"I'm convinced by the data showing that FOXO is required for TkRNAi to prevent lifespan shortening by Ap, but FOXO doesn't only respond to insulin signalling and can't be taken by itself to indicate a role for insulin signalling which the authors appear to do here."

To the best of my knowledge, Foxo has only been shown to be required for lifespan extension/modulation by a reduction in insulin-like signalling. I.e. it does respond to other pathways but this is the only one where Foxo activity is known to modulate lifespan.

Reviewer 3

I agree with reviewer 1 that point raised under (1) does not appear strictly required for the conclusions of the manuscript.

Both reviewers 1 and 3:

I have a different take on the results of experiments where IPCs are manipulated. To me, Figure 7D and E show that ablating the IPCs removes the difference between Ax and Ap i.e. the IPCs are involved and insulin-like signalling is likely involved. The fact that RNAi against the TKR99D receptor does not have the same effect, does not matter (the sensing could happen in different neurons). Similarly, dilp expression is only a minor readout of what is happening with insulin-like signalling - dilps are controlled at the level of secretion.

However, I would be happy for the authors to present different arguments and make a reasonable conclusion, which may differ from mine. But I think the arguments I present above should be taken into account.

Significance

The main contribution of this manuscript is the identification of a mechanism that links the microbiota to lifespan. This is very exciting and topical for several reasons:

1) The microbiota is very important for overall health but it is still unclear how. Studying the interaction between microbiota and health is an emerging, growing field, and one that has attracted a lot of interest, but one that is often lacking in mechanistic insight. Identifying mechanisms provides opportunities for therapies. The main impact of this study comes from using the fruit fly to identify a mechanism.

2) It is very interesting that the authors focus on an endocrine mechanism, especially with the clear clinical relevance of gut hormones to human health recently demonstrated with new, effective therapies (e.g. Wegovy).

3) Tk is emerging as an important fly hormone and this study adds a new and interesting dimension by placing TK between microbiota and lifespan.

I think the manuscript will be of great interest to researchers in ageing, human and animal physiology and in gut endocrinology and gut function.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary:

In this study the authors use a Drosophila model to demonstrate that Tachykinin (Tk) expression is regulated by the microbiota. In Drosophila conventionally reared (CR) flies are typically shorter lived than those raised without a microbiota (axenic). Here, knockdown of Tk expression is found to prevent lifespan shortening by the microbiota and the reduction of lipid stores typically seen in CR flies when compared to axenic counterparts. It does so without reducing food intake or fecundity which are often seen as necessary trade-offs for lifespan extension. Further, the strength of the interaction between Tk and the microbiota is found to be bacteria specific and is stronger in Acetobacter pomorum (Ap) monoassociated flies compared to Levilactobacillus brevis (Lb) monoassociation. The impact on lipid storage was also only apparent in Ap-flies. Building on these findings the authors show that gut specific knockdown is largely sufficient to explain these phenotypes. Knockdown of the Tk receptor, TkR99D, in neurons recapitulates the lifespan phenotype of intestinal Tk knockdown supporting a model whereby Tk from the gut signals to TkR99D expressing neurons to regulate lifespan. In addition, the authors show that FOXO may have a role in lifespan regulation by the Tk-microbiota interaction. However, they rule out a role for insulin producing cells or Akh-producing cells suggesting the microbiota-Tk interaction regulates lifespan through other, yet unidentified, mechanisms.

Major comments:

Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. In particular, the impact of TkRNAi on lifespan and lipid levels, the central finding in this study, has been demonstrated multiple times in different experiments and using different genetic tools. As a result, I don't feel that additional experimental work is necessary to support the current conclusions. However, I find it hard to assess the robustness of the lifespan data from the other manipulations used (TkR99DRNAi, TkRNAi in dFoxo mutants etc.) because information on the population size and whether these experiments have been replicated is lacking. Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done? For all other data it is clear how many replicates have been done, and the methods give enough detail for all experiments to be reproduced.

Minor comments:

While I feel the conclusions of this study are well supported by the data I found this to be a complex read and in places hard to follow. I feel some work is necessary in the writing to help the reader follow the authors logic. Below I describe some of the issues that confused me and provide some suggestions that I hope the authors will find helpful.

Survival curves The authors state that the lifespan difference between CR and axenic flies disappears with TkRNAi because TkRNAi CR flies are longer lived, rather than because TkRNAi axenic flies are shorter lived. Is this consistent in every TkRNAi experiment? It's hard for the reader to assess this because the relevant lifespan curves are presented on separate plots. I'd encourage the authors to provide lifespan plots that enable comparison between all conditions. For example, in figures 2 and 6 the reader wants to directly compare between RU- and RU+ but can't easily do so. Additional plots could be made available in the supplementary figures showing the comparisons that are not easy to make on the main figures.

Consistent framing of the data Do the interventions shorten lifespan relative to the axenic cohort? Or do they prevent lifespan extension by axenic conditions? Both statements are valid, and the authors need to be consistent in which one they use to avoid confusing the reader. For example, line 325 says TkR86CRNAi prevents lifespan extension in axenic flies. Given the framing in the previous sections, it might be clearer to say that TkR86CRNAi shortens the lifespan of axenic flies to that of CR flies in contrast to TkRNAi and TkR99DRNAi which don't.

The impact of TkRNAi on lipid levels in axenic flies TkRNAi consistently reduces lipid levels in axenic flies (Figs 2E, 3D), essentially phenocopying the loss of lipid stores seen in control conventionally reared (CR) flies relative to control axenic. This suggests that the previously reported role of Tk in lipid storage - demonstrated through increased lipid levels in TkRNAi flies (Song et al (2014) Cell Rep 9(1): 40) - is dependent on the microbiota. In the absence of the microbiota TkRNAi reduces lipid levels. The lack of acknowledgement of this in the text is confusing for the reader because it is inconsistent with the microbiota driving both higher Tk expression and higher lipid storage. If the microbiota increases Tk expression and this results in reduced lipid storage, why does reduced Tk expression also result in reduced lipid storage in axenic flies? This could further highlight the unique impact that the interaction between TkRNAi and the microbiota has on lipid storage, given it reverses both the impact of the microbiota alone and TkRNAi alone. I feel this aspect of the data should be given more attention in the text both for clarity and because it may be telling us something important about the function of Tk. The current framing around pleiotropic effects is valid, and the impact of Tk on lipid storage is clearly independent of its impact on lifespan and so is not central to this study. However, I feel a short additional paragraph to acknowledge this nuance of the data is needed. It can be made clear in the text that further exploration is beyond the scope of the current study.

Role of insulin signalling and insulin producing cells I'm convinced by the data showing that FOXO is required for TkRNAi to prevent lifespan shortening by Ap, but FOXO doesn't only respond to insulin signalling and can't be taken by itself to indicate a role for insulin signalling which the authors appear to do here.

I would expect ablation of IPCs to have the opposite effect to foxo mutation and to increase FOXO activity throughout the organism due to a reduction in Dilp levels and so reduced insulin signalling. So, I have struggled to follow the authors logic in ablating the IPCs and feel a clear statement on what they expected the outcome to be would help the reader. They find that TkRNAi still prevents lifespan shortening by Ap when IPCs are ablated and that TkR99DRNAi in IPCs also doesn't block lifespan shortening by Ap despite reducing the expression of dilp3 and dilp5. To me these data rule out a role for insulin signalling despite the requirement for FOXO and yet the authors conclude that insulin signalling is involved in the response to Ap and TkRNAi, although not obligately (lines 420 - 422 and 511 - 512). Can the authors clarify their logic in concluding a role for insulin signalling, and qualify this conclusion with appropriate consideration of alternative hypotheses? The potential involvement of other signalling inputs to FOXO activity, e.g. immune signalling and JNK, should be acknowledged and warrants some discussion.

Typographical errors:

Incomplete sentence line 121 to 122 - starting "Cox proportional hazards.... and posthoc tests (Fig 2b).

Line 123 "EMMs" - define abbreviation on first use

References to Fig 2b (first given on line 122), should be capitalised to Fig 2B for consistency.

Lines 231 and 317 - the phrase "steady state (microbiota independent) expression" in reference to flyATLAS 2 data could be misleading. The term "microbiota independent" could suggest that expression levels have been shown not to be regulated by the microbiota and this is not the case. The authors should change this to simply state they are referring to steady state expression in conventionally reared flies.

Referees cross-commenting

Below are brief comments on the revision suggestions that reviewers 2 and 3 have requested.

Reviewer 2

1. I agree that confirmation that TkRNAi doesn't impact microbial levels could be helpful and would be straightforward for the authors to do. However, I don't feel it's essential to support the central claims of the paper.
2. I agree.
3. I don't feel that any of these experiments supports a role for insulin signalling, so I don't feel that this additional control is needed.
4. It would be a good addition to have lifespan data from a separate knockdown line for corroboration. However, this has already been done in several different genetic backgrounds through crosses with different driver lines in multiple tissues, so I feel it's unnecessary given the time and resources that lifespan experiments take. There's also the caveat that different RNAi lines can knockdown to different extents so that would have to be assessed as well and if there's a difference it may mean that ultimately not much can be concluded from this additional experiment.
5. A good suggestion, but not straightforward and depends on the availability of the necessary tools, or possibly the generation of new tools. One for a follow up study.
6. I feel this is not important enough to the central findings of the study to warrant the extra work.
7. I agree.

Reviewer 3 1. Imaging calcium signalling is not straightforward unless a lab already has the tools available and optimised. If Tk+ EEs show changes in calcium signalling I'm not convinced that this tells us anything specific to the Tk-microbiota interaction. The point is the role of Tk itself, not the broader activity of the cells that express it. 2. I agree this needs clarification. 3. I agree that this would add depth, if feasible, but feel it's not essential to support the current conclusions. 4. This is a minor point and given the RT-qPCR data and the RNAseq data corroborate each other I'm convinced that Tk levels are elevated. 5. I feel exploring this in males is opening an additional line of enquiry beyond the scope of the current study. Either the phenotypes are the same - in which case what is added? - or they are different but there's no scope to assess why. A good suggestion for a follow up study. 6. No comment. 7. Agreed.

One final comment. It's true that FOXO has only been shown to regulate lifespan in the context of insulin signalling. However, as far as I'm aware it hasn't been shown not to regulate lifespan downstream of it's other activators, this simply hasn't been explored due to the historical focus on insulin signalling in this field. In the context of host-microbiota interactions considering other pathways the activate FOXO, such as immune and JNK signals, would make sense.

Reviewed by Dr Rebecca Clark, Department of Biosciences, Durham University

Significance

Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. We have known that the microbiota influence lifespan for some time but the mechanisms by which they do so have remained elusive. This study identifies one such mechanism and as a result opens several avenues for further research. The Tk-microbiota interaction is shown to be important for both lifespan and lipid homeostasis, although it's clear these are independent phenotypes. The fact that the outcome of the Tk-microbiota interaction depends on the bacterial species is of particular interest because it supports the idea that manipulation of the microbiota, or specific aspects of the host-microbiota interaction, may have therapeutic potential.<br /> These findings will be of interest to a broad readership spanning host-microbiota interactions and their influence on host health. They move forward the study of microbial regulation of host longevity and have relevance to our understanding of microbial regulation of host lipid homeostasis. They will also be of significant interest to those studying the mechanisms of action and physiological roles of Tachykinins.

Field of expertise: Drosophila, gut, ageing, microbiota, innate immunity

eLife Assessment

This work provides one of the first important attempts to look at Drosophila immune responses against bacterial, viral, and fungal pathogens in a way that combines the roles of four major arms in immunity (Imd signaling, Toll signaling, phagocytosis, and melanization) rather than studying them separately. The findings are compelling and the tools provided can be used as they are, or built upon, in various contexts.

Reviewer #1 (Public review):

Summary:

The innate immune system serves as the first line of defense against invading pathogens. Four major immune-specific modules-the Toll pathway, the Imd pathway, melanization, and phagocytosis-play critical roles in orchestrating the immune response. Traditionally, most studies have focused on the function of individual modules in isolation. However, in recent years, it has become increasingly evident that effective immune defense requires intricate interactions among these pathways.

Despite this growing recognition, the precise roles, timing, and interconnections of these immune modules remain poorly understood. Moreover, addressing these questions represents a major scientific undertaking.

Strengths:

In this manuscript, Ryckebusch et al. systematically evaluate both the individual and combined contributions of these four immune modules to host defense against a range of pathogens. Their findings significantly enhance our understanding of the layered architecture of innate immunity.

Reviewer #2 (Public review):

Summary:

In this work, the authors take a holistic view at the Drosophila immunity by selecting four major components of fly immunity often studied separately (Toll signaling, Imd signaling, phagocytosis and melanization), and studying their combinatory effects on the efficiency of the immune response. They achieve this by using fly lines mutant for one of these components, or modules, as well as for a combination of them, and testing the survival of these flies upon infection with a plethora of pathogens (bacterial, viral and fungal).

Strengths:

It is clear that this manuscript has required a large amount of hands-on work, considering the number of pathogens, mutations and timepoints tested. In my opinion, this work is a very welcome addition to the literature on fly immune responses, which obviously do not occur one type of a response at a time, but in parallel, subsequently and/or are interconnected. I find that the major strength of this work is the overall concept, which is made possible by the mutations designed to target the specific immune function of each module, without effects on other functions. I believe that the combinatory mutants will be of use for the fly community and enable further studies of interplay of these components of immune response in various settings.

To control for the effects arising from the genetic variation other than the intended mutations, the mutants have been backcrossed into a widely used, isogenized Drosophila strain called w1118. Therefore, the differences accounted for by the genotype are controlled.

I also appreciate that the authors have investigated the two possible ways of dealing with an infection: tolerance and resistance, and how the modules play into those.

Weaknesses:

While controlling for the background effects is vital, the w1118 background is problematic (an issue not limited to this manuscript) because of the wide effects of the white mutation on several phenotypes (also other than eye color/eyesight). It is a possibility that the mutation influences the functionality of the immune response components. I acknowledge that it is not reasonable to ask for data in different backgrounds better representing a "wild type" fly, but I think this matter should be brought up and discussed.

The whole study has been conducted on male flies. Immune responses show quite extensive sex-specific variation across a variety of species studied, also in the fly. But the reasons for this variation are not fully understood. Therefore, I suggest that the authors would conduct a subset of experiments on female flies to see if the findings apply to both sexes, especially the infection-specificity of the module combinations.

Comments on the revised manuscript:

I appreciate the author's responses to the points I raised and the additional work they have conducted. The authors have now discussed the possible background effect and added an experiment on female flies showing that the module function is applicable to both sexes.

hes not filthy :(

I'm somehow missing the transition from general networks to the networks of charging stations. Should we consider the charging station networks on this level at all, or better to handle it as an use case example?

Is the meaning "gateway shows" or "the section "Mobile radio gateway" shows"?

A long dash would be better. Or at least it have to be used consistently within the document.

Should here be a period at the end of each point? It should be at least consistent.

taxnegative = distortionary taxes and unproductive spending, or nondistortionary taxes, lowers growth -0.18

taxambigous = lower -0.11

taxpositive = 0.02 growtyh

taxpositive = positive economic growth. taxnegative = negative growth. this is the theory on what forms of taxation create this

publication bias

findings

math

growth tax effect

big idea

gun up unemploymet dgaf. gun up investment down

gun up investment down

gun up gdp down a little bit

military dont afect unemploument

nongun up unemployment down

nongun up productive up

guns up private investment down

contemporarous equation math

holy shit thats math

data

guns employ

guns butter math

guns butter

defense-growth chart

supply side

demand side

Taney points to old state laws that treated Black people unfairly and says this proves they were never meant to be citizens. He’s using discrimination as evidence. I disagree because laws that were unjust shouldn’t be used to defend injustice. They show racism, not truth.

Taney claims that since enslaved Africans weren’t considered “the people” back then, they can’t be citizens now. He’s using historical exclusion as proof. I strongly disagree because “all men are created equal” should apply to everyone. Just because people were wrong in the past doesn’t mean we should keep those ideas alive.

Taney looks back to colonial times to decide who counts as a citizen. He uses old laws and traditions to prove his point, which is exactly how originalists think. I disagree because those old systems supported slavery. Using them as a guide for freedom is unfair and outdated.

Here, Taney says judges have to follow what the Constitution meant when it was first written. He doesn’t believe it should change with time or new ideas. I disagree because that way of thinking keeps the country stuck in the past. The Constitution should grow with society and reflect fairness for everyone.

Taney is asking if Black people can ever be part of the “political community” that the founders created. He basically says that since the people who wrote the Constitution didn’t see them as citizens, they still aren’t. I disagree because Taney uses racist beliefs from the 1700s to decide what’s right now. The founders’ views shouldn’t stop people from being treated equally today.

rhythm for euphony created with alliteration

If the institutions are effective - is there enough motivation to create informal mechanisms for addressing "gaps"? If actors are mindful of the sanctions for transgressions - aren't informal institutions also not those transgressors?

It's not only about the result - it's about what gets knocked over and broken in the process of reaching the result

For Nihilism, I find it to be a fascinating and controversial ethical framework. I understand some people prefer this theory more (even I believed this theory when I was a teenager), because in the end of life everything is gone, nothing matters in the end. But what I believe is that we create meaning through what we choose to value. So while Nihilism' core is objective morality, it might indirectly encourages personal responsibility.

Of course, sometimes planning is about not repeating past mistakes.

so maybe never tell your ideas to these types of people in the first place ? you share your ideas and goals with others, you often end up not achieving them. if you tell your ideas to common people, you should be prepared for this kind of reaction from them. personally, if a well educated and well known person in the industry or the community said that to me, i would probably accept it and drop the idea.

One thing that really stood out to me in this chapter was the discussion about how social networking platforms evolved from early services like Friendster and MySpace to the dominant platforms we know today, like Facebook and YouTube. It made me think about how quickly digital trends shift and how easily one platform can completely overtake another. Personally, I remember having a MySpace account in middle school, and it was a big part of how we socialized online. But within just a few years, everyone moved to Facebook, and MySpace basically disappeared. It makes me wonder: will the same thing happen to today's dominant platforms like Instagram or TikTok? Are they just temporary until the next big shift happens?.

I find this passage particularly interesting because it reminds me how similar our current use of social media is to the original concept of blogging. People initially treated websites as “diaries” for documenting life and sharing thoughts. While today's platforms offer more powerful features, their core purpose remains self-expression and connecting with others. This also illustrates how the internet has evolved incrementally—from simple personal journals to today's complex social networks—reflecting humanity's enduring pursuit of communication and connection.

In my English class we were required to read this article called "The I in Internet". It talked about how the new wave of social media, Web 2.0, prioritized individuals and one's self. It turned into a community where everyone started portraying their best selves. It is interesting to see how social media has changed overtime, as it was once seen as much more positive entity then it is now.

Here is a link to the article: https://lab.cccb.org/en/the-i-in-the-internet/

DOI: 10.3390/v17101318

Resource: Pennsylvania State University Huck Institutes of the Life Sciences Genomics Core Facility (RRID:SCR_023645)

Curator: @scibot

SciCrunch record: RRID:SCR_023645

What is this?

.to-page-note

.from - https://hyp.is/r3EV6qQgEfCfF7cH5XZZfQ/www.google.com/search?q=develop+new+software+how+is+the+book+coming+along&oq=develop+new+software+how+is+the+book+coming+along

DOI: 10.1016/j.cmet.2025.09.003

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:008231

What is this?

Returns the integer remainder of dividing the two operands.

Ex: 12 % 5 returns 2

Here we can state that the client doesn't verify the match, it's the admin who must ensure this.

I found the section about early email communication really interesting. It’s fascinating to think that something as simple as sending messages back and forth was once considered a major technological advancement.

With the internet and social media development, various kinds of platforms and apps start to emerge. By reading through this unit, I'm so glad to see and feel the development of social media, which made the communication between people so easy and convenient. Meanwhile, So many bad information starts to appear on the internet. This is an uncontrollable thing. So all we can do is filtrate the information from the internet and choose what is beneficial for us.

Genuinely mindblowing name. I thought this story would be a singular instance, but the twist that it ended up being the massive platform we know as 4chan. Is the point of social media to allow complete and unrestricted socialization, or something else entirely? The point I'm trying to make and I think the major takeaway we can glean from 4chan now that it's a few years removed, is that complete lack of restriction on the internet usually serves to enable people to engage is violent or degenerate behaviors with significantly less consequences than there would be in the real world.

This makes me wonder, should we have restrictions on what can be posted on social media? Isn't that taking away freedom of speech? Is it the CEO's responsibility to ensure that the social media site is being used for good? Does the CEO carry that burden, or is it on the individual users?

جيمع Laravel Config موجدوة داخل مجلد Config وكل هذه موثقة جيدا في doc

The sound of Silence

looked up at the stars and gazed at the moon. Because there are many cycles that exist within the cycles of the seasons, like the cycle of the moon and the cycle of the stars. And we understood these relationships because they were the most foundational forms of the ways we lived our lives. Not distanced, but in constant relationship, constant attunement, constant attentiveness to the variation of how the seasons expresses itself in form, in the circular nature of existence. Each day, each year, we observed this. We participated in this.

controlled master

instead of participants

eLife Assessment

This potentially valuable study presents claims of evidence for coordinated membrane potential oscillations in E. coli biofilms that can be linked to a putative K+ channel and that may serve to enhance photo-protection. The finding of waves of membrane potential would be of interest to a wide audience from molecular biology to microbiology and physical biology. Unfortunately, a major issue is that it is unclear whether the dye used can act as a Nernstian membrane potential dye in E. coli. The arguments of the authors, who largely ignore previously published contradictory evidence, are not adequate in that they do not engage with the fact that the dye behaves in their hands differently than in the hands of others. In addition, the lack of proper validation of the experimental method including key control experiments leaves the evidence incomplete.

Reviewer #1 (Public Review):

(1) Significance of the findings:

Cell-to-cell communication is essential for higher functions in bacterial biofilms. Electrical signals have proven effective in transmitting signals across biofilms. These signals are then used to coordinate cellular metabolisms or to increase antibiotic tolerance. Here, the authors have reported for the first time coordinated oscillation of membrane potential in E. coli biofilms that may have a functional role in photoprotection.

(2) Strengths of the manuscript:

- The authors report original data.<br /> - For the first time, they showed that coordinated oscillations in membrane potential occur in E. Coli biofilms.<br /> - The authors revealed a complex two-phase dynamic involving distinct molecular response mechanisms.<br /> - The authors developed two rigorous models inspired by 1) Hodgkin-Huxley model for the temporal dynamics of membrane potential and 2) Fire-Diffuse-Fire model for the propagation of the electric signal.<br /> - Since its discovery by comparative genomics, the Kch ion channel has not been associated with any specific phenotype in E. coli. Here, the authors proposed a functional role for the putative gated-voltage-gated K+ ion channel (Kch channel) : enhancing survival under photo-toxic conditions.

(3) Weakness:

- Contrarily to what is stated in the abstract, the group of B. Maier has already reported collective electrical oscillations in the Gram-negative bacterium Neisseria gonorrhoeae (Hennes et al., PLoS Biol, 2023).<br /> - The data presented in the manuscript are not sufficient to conclude on the photo-protective role of the Kch channel. The authors should perform the appropriate control experiments related to Fig4D,E, i.e. reproduce these experiments without ThT to rule out possible photo-conversion effects on ThT that would modify its toxicity. In addition, it looks like the data reported on Fig 4E are extracted from Fig 4D. If this is indeed the case, it would be more conclusive to report the percentage of PI-positive cells in the population for each condition. This percentage should be calculated independently for each replicate. The authors should then report the average value and standard deviation of the percentage of dead cells for each condition.<br /> - Although Fig 4A clearly shows that light stimulation has an influence on the dynamics of ThT signal in the biofilm, it is important to rule out possible contributions of other environmental variations that occur when the flow is stopped at the onset of light stimulation. I understand that for technical reasons, the flow of fresh medium must be stopped for the sake of imaging. Therefore, I suggest to perform control experiments consisting in stopping the flow at different time intervals before image acquisition (30min or 1h before). If there is no significant contribution from environmental variations due to medium perfusion arrest, the dynamics of ThT signal must be unchanged regardless of the delay between flow stop and the start of light stimulation.<br /> - To precise the role of K+ in the habituation response, I suggest using the ionophore valinomycin at sub-inhibitory concentrations (5 or 10µM). It should abolish the habituation response. In addition, the Kch complementation experiment exhibits a sharp drop after the first peak but on a single point. It would be more convincing to increase the temporal resolution (1min->10s) to show that there are indeed a first and a second peak. Finally, the high concentration (100µM) of CCCP used in this study completely inhibits cell activity. Therefore, it is not surprising that no ThT dynamics was observed upon light stimulation at such concentration of CCCP.<br /> - Since TMRM signal exhibits a linear increase after the first response peak (Supp Fig1D), I recommend to mitigate the statement at line 78.<br /> - Electrical signal propagation is an important aspect of the manuscript. However, a detailed quantitative analysis of the spatial dynamics within the biofilm is lacking. At minima, I recommend to plot the spatio-temporal diagram of ThT intensity profile averaged along the azimuthal direction in the biofilm. In addition, it is unclear if the electrical signal propagates within the biofilm during the second peak regime, which is mediated by the Kch channel: I have plotted the spatio-temporal diagram for Video S3 and no electrical propagation is evident at the second peak. In addition, the authors should provide technical details of how R^2(t) is measured in the first regime (Fig 7E).<br /> - In the series of images presented in supplementary Figure 4A, no wavefront is apparent. Although the microscopy technics used in this figure differs from other images (like in Fig2), the wavefront should be still present. In addition, there is no second peak in confocal images as well (Supp Fig4B) .<br /> - Many important technical details are missing (e.g. biofilm size, R^2, curvature and 445nm irradiance measurements). The description of how these quantitates are measured should be detailed in the Material & Methods section.<br /> - Fig 5C: The curve in Fig 5D seems to correspond to the biofilm case. Since the model is made for single cells, the curve obtained by the model should be compared with the average curve presented in Fig 1B (i.e. single cell experiments).<br /> - For clarity, I suggest to indicate on the panels if the experiments concern single cell or biofilm experiments. Finally, please provide bright-field images associated to ThT images to locate bacteria.<br /> - In Fig 7B, the plateau is higher in the simulations than in the biofilm experiments. The authors should add a comment in the paper to explain this discrepancy.

Reviewer #2 (Public Review):

The authors use ThT dye as a Nernstian potential dye in E. coli. Quantitative measurements of membrane potential using any cationic indicator dye are based on the equilibration of the dye across the membrane according to Boltzmann's law.

Ideally, the dye should have high membrane permeability to ensure rapid equilibration. Others have demonstrated that E.coli cells in the presence of ThT do not load unless there is blue light present, that the loading profile does not look like it is expected for a cationic Nernstian dye. They also show that the loading profile of the dye is different for E.coli cells deleted for the TolC pump. I, therefore, objected to interpreting the signal from the ThT as a Vm signal when used in E.coli. Nothing the authors have said has suggested that I should be changing this assessment.

Specifically, the authors responded to my concerns as follows:

(1) 'We are aware of this study, but believe it to be scientifically flawed. We do not cite the article because we do not think it is a particularly useful contribution to the literature.' This seems to go against ethical practices when it comes to scientific literature citations. If the authors identified work that handles the same topic they do, which they believe is scientifically flawed, the discussion to reflect that should be included.

(2)'The Pilizota group invokes some elaborate artefacts to explain the lack of agreement with a simple Nernstian battery model. The model is incorrect not the fluorophore.'<br /> It seems the authors object to the basic principle behind the usage of Nernstian dyes. If the authors wish to use ThT according to some other model, and not as a Nernstian indicator, they need to explain and develop that model. Instead, they state 'ThT is a Nernstian voltage indicator' in their manuscript and expect the dye to behave like a passive voltage indicator throughout it.

(3)'We think the proton effect is a million times weaker than that due to potassium i.e. 0.2 M K+<br /> versus 10-7 M H+. We can comfortably neglect the influx of H+ in our experiments.'<br /> I agree with this statement by the authors. At near-neutral extracellular pH, E.coli keeps near-neutral intracellular pH, and the contribution from the chemical concentration gradient to the electrochemical potential of protons is negligible. The main contribution is from the membrane potential. However, this has nothing to do with the criticism to which this is the response of the authors. The criticism is that ThT has been observed not to permeate the cell without blue light. The blue light has been observed to influence the electrochemical potential of protons (and given that at near-neutral intracellular and extracellular pH this is mostly the membrane potential, as authors note themselves, we are talking about Vm effectively). Thus, two things are happening when one is loading the ThT, not just expected equilibration but also lowering of membrane potential. The electrochemical potential of protons is coupled via the membrane potential to all the other electrochemical potentials of ions, including the mentioned K+.

(4) 'The vast majority of cells continue to be viable. We do not think membrane damage is dominating.' In response to the question on how the authors demonstrated TMRM loading and in which conditions (and while reminding them that TMRM loading profile in E.coli has been demonstrated in Potassium Phosphate buffer). The request was to demonstrate TMRM loading profile in their condition as well as to show that it does not depend on light. Cells could still be viable, as membrane permeabilisation with light is gradual, but the loading of ThT dye is no longer based on simple electrochemical potential (of the dye) equilibration.

(5) On the comment on the action of CCCP with references included, authors include a comment that consists of phrases like 'our understanding of the literature' with no citations of such literature. Difficult to comment further without references.

(6) 'Shielding would provide the reverse effect, since hyperpolarization begins in the dense centres of the biofilms. For the initial 2 hours the cells receive negligible blue light. Neither of the referee's comments thus seem tenable.'<br /> The authors have misunderstood my comment. I am not advocating shielding (I agree that this is not it) but stating that this is not the only other explanation for what they see (apart from electrical signaling). The other I proposed is that the membrane has changed in composition and/or the effective light power the cells can tolerate. The authors comment only on the light power (not convincingly though, giving the number for that power would be more appropriate), not on the possible changes in the membrane permeability.

(7) 'The work that TolC provides a possible passive pathway for ThT to leave cells seems slightly niche. It just demonstrates another mechanism for the cells to equilibrate the concentrations of ThT in a Nernstian manner i.e. driven by the membrane voltage.' I am not sure what the authors mean by another mechanism. The mechanism of action of a Nernstian dye is passive equilibration according to the electrochemical potential (i.e. until the electrochemical potential of the dye is 0).

(8) 'In the 70 years since Hodgkin and Huxley first presented their model, a huge number of similar models have been proposed to describe cellular electrophysiology. We are not being hyperbolic when we state that the HH models for excitable cells are like the Schrödinger<br /> equation for molecules. We carefully adapted our HH model to reflect the currently understood electrophysiology of E. coli.'

I gave a very concrete comment on the fact that in the HH model conductivity and leakage are as they are because this was explicitly measured. The authors state that they have carefully adopted their model based on what is currently understood for E.coli electrophysiology. It is not clear how. HH uses gKn^4 based on Figure2 here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1392413/pdf/jphysiol01442-0106.pdf, i.e. measured rise and fall of potassium conductance on msec time scales. I looked at the citation the authors have given and found a resistance of an entire biofilm of a given strain at 3 applied voltages. So why n^4 based on that? Why does unknown current have gqz^4 form? Sodium conductance in HH is described by m^3hgNa (again based on detailed conductance measurements), so why unknown current in E.coli by gQz^4? Why leakage is in the form that it is, based on what measurement?

Throughout their responses, the authors seem to think that collapsing the electrochemical gradient of protons is all about protons, and this is not the case. At near neutral inside and outside pH, the electrochemical potential of protons is simply membrane voltage. And membrane voltage acts on all ions in the cell.

Authors have started their response to concrete comments on the usage of ThT dye with comments on papers from my group that are not all directly relevant to this publication. I understand that their intention is to discredit a reviewer but given that my role here is to review this manuscript, I will only address their comments to the publications/part of publications that are relevant to this manuscript and mention what is not relevant.

Publications in the order these were commented on.

(1) In a comment on the paper that describes the usage of ThT dye as a Nernstian dye authors seem to talk about a model of an entire active cell.<br /> 'Huge oscillations occur in the membrane potentials of E. coli that cannot be described by the SNB model.' The two have nothing to do with each other. Nernstian dye equilibrates according to its electrochemical potential. Once that happens it can measure the potential (under the assumption that not too much dye has entered and thus lowered too much the membrane potential under measurement). The time scale of that is important, and the dye can only measure processes that are slower than that equilibration. If one wants to use a dye that acts under a different model, first that needs to be developed, and then coupled to any other active cell model.

(2) The part of this paper that is relevant is simply the usage of TMRM dye. It is used as Nernstian dye, so all the above said applies. The rest is a study of flagellar motor.

(3) The authors seem to not understand that the electrochemical potential of protons is coupled to the electrochemical potentials of all other ions, via the membrane potential. In the manuscript authors talk about, PMF~Vm, as DeltapH~0. Other than that this publication is not relevant to their current manuscript.

(4) The manuscript in fact states precisely that PMF cannot be generated by protons only and some other ions need to be moved out for the purpose. In near neutral environment it stated that these need to be cations (K+ e.g.). The model used in this manuscript is a pump-leak model. Neither is relevant for the usage of ThT dye.

Further comments include, along the lines of:

'The editors stress the main issue raised was a single referee questioning the use of ThT as an indicator of membrane potential. We are well aware of the articles by the Pilizota group and we believe them to be scientifically flawed. The authors assume there are no voltage-gated ion channels in E. coli and then attempt to explain motility data based on a simple Nernstian battery model (they assume E. coli are unexcitable<br /> matter). This in turn leads them to conclude the membrane dye ThT is faulty, when in fact it is a problem with their simple battery model.'

The only assumption made when using a cationic Nernstian dye is that it equilibrates passively across the membrane according to its electrochemical potential. As it does that, it does lower the membrane potential, which is why as little as possible is added so that this is negligible. The equilibration should be as fast as possible, but at the very least it should be known, as no change in membrane potential can be measured that is faster than that.

This behaviour should be orthogonal to what the cell is doing, it is a probe after all. If the cell is excitable, a Nernstian dye can be used, as long as it's still passively equilibrating and doing so faster than any changes in membrane potential due to excitations of the cells. There are absolutely no assumptions made on the active system that is about to be measured by this expected behaviour of a Nernstian dye. And there shouldn't be, it is a probe. If one wants to use a dye that is not purely Nernstian that behaviour needs to be described and a model proposed. As far as I can find, authors do no such thing.

There is a comment on the use of a flagellar motor as a readout of PMF, stating that the motor can be stopped by YcgR citing the work from 2023. Indeed, there is a range of references such as https://doi.org/10.1016/j.molcel.2010.03.001 that demonstrate this (from around 2000-2010 as far as I am aware). The timescale of such slowdown is hours (see here Figure 5 https://www.cell.com/cell/pdf/S0092-8674(10)00019-X.pdf). Needless to say, the flagellar motor when used as a probe, needs to stay that in the conditions used. Thus one should always be on the lookout at any other such proteins that could slow it down and we are not aware of yet or make the speed no longer proportional to the PMF. In the papers my group uses the motor the changes are fast, often reversible, and in the observation window of 30min. They are also the same with DeltaYcgR strain, which we have not included as it seemed given the time scales it's obvious, but certainly can in the future (as well as stay vigilant on any conditions that would render the motor a no longer suitable probe for PMF).

Reviewer #3 (Public Review):

This manuscript by Akabuogu et al. investigates membrane potential dynamics in E. coli. Membrane potential fluctuations have been observed in bacteria by several research groups in recent years, including in the context of bacterial biofilms where they have been proposed to play a role in cellular communication. Here, these authors investigate membrane potential in E. coli, in both single cells and biofilms. I have reviewed the revised manuscript provided by the authors, as well as their responses to the initial reviews; my opinion about the manuscript is largely unchanged. I have focused my public review on those issues that I believe to be most pressing, with additional comments included in the review to authors. Although these authors are working in an exciting research area, the evidence they provide for their claims is inadequate, and several key control experiments are still missing. In some cases, the authors allude to potentially relevant data in their responses to the initial reviews, but unfortunately these data are not shown. Furthermore, I cannot identify any traveling wavefronts in the data included in this manuscript. In addition to the challenges associated with the use of Thioflavin-T (ThT) raised by the second reviewer, these caveats make the work presented in this manuscript difficult to interpret.

First, some of the key experiments presented in the paper lack required controls:

(1) This paper asserts that the observed ThT fluorescence dynamics are induced by blue light. This is a fundamental claim in the paper, since the authors go on to argue that these dynamics are part of a blue light response. This claim must be supported by the appropriate negative control experiment measuring ThT fluorescence dynamics in the absence of blue light- if this idea is correct, these dynamics should not be observed in the absence of blue light exposure. If this experiment cannot be performed with ThT since blue light is used for its excitation, TMRM can be used instead.

In response to this, the authors wrote that "the fluorescent baseline is too weak to measure cleanly in this experiment." If they observe no ThT signal above noise in their time lapse data in the absence of blue light, this should be reported in the manuscript- this would be a satisfactory negative control. They then wrote that "It appears the collective response of all the bacteria hyperpolarization at the same time appears to dominate the signal." I am not sure what they mean by this- perhaps that ThT fluorescence changes strongly only in response to blue light? This is a fundamental control for this experiment that ought to be presented to the reader.

(2) The authors claim that a ∆kch mutant is more susceptible to blue light stress, as evidenced by PI staining. The premise that the cells are mounting a protective response to blue light via these channels rests on this claim. However, they do not perform the negative control experiment, conducting PI staining for WT the ∆kch mutant in the absence of blue light. In the absence of this control it is not possible to rule out effects of the ∆kch mutation on overall viability and/or PI uptake. The authors do include a growth curve for comparison, but planktonic growth is a very different context than surface-attached biofilm growth. Additionally, the ∆kch mutation may have impacts on PI permeability specifically that are not addressed by a growth curve. The negative control experiment is of key importance here.

Second, the ideas presented in this manuscript rely entirely on analysis of ThT fluorescence data, specifically a time course of cellular fluorescence following blue light treatment. However, alternate explanations for and potential confounders of the observed dynamics are not sufficiently addressed:

(1) Bacterial cells are autofluorescent, and this fluorescence can change significantly in response to stress (e.g. blue light exposure). To characterize and/or rule out autofluorescence contributions to the measurement, the authors should present time lapse fluorescence traces of unstained cells for comparison, acquired under the same imaging conditions in both wild type and ∆kch mutant cells. In their response to reviewers the authors suggested that they have conducted this experiment and found that the autofluorescence contribution is negligible, which is good, but these data should be included in the manuscript along with a description of how these controls were conducted.

(2) Similarly, in my initial review I raised a concern about the possible contributions of photobleaching to the observed fluorescence dynamics. This is particularly relevant for the interpretation of the experiment in which catalase appears to attenuate the decay of the ThT signal; this attenuation could alternatively be due to catalase decreasing ThT photobleaching. In their response, the authors indicated that photobleaching is negligible, which would be good, but they do not share any evidence to support this claim. Photobleaching can be assessed in this experiment by varying the light dosage (illumination power, frequency, and/or duration) and confirming that the observed fluorescence dynamics are unaffected.

Third, the paper claims in two instances that there are propagating waves of ThT fluorescence that move through biofilms, but I do not observe these waves in any case:

(1) The first wavefront claim relates to small cell clusters, in Fig. 2A and Video S2 and S3 (with Fig. 2A and Video S2 showing the same biofilm.) I simply do not see any evidence of propagation in either case- rather, all cells get brighter and dimmer in tandem. I downloaded and analyzed Video S3 in several ways (plotting intensity profiles for different regions at different distances from the cluster center, drawing a kymograph across the cluster, etc.) and in no case did I see any evidence of a propagating wavefront. (I attempted this same analysis on the biofilm shown in Fig. 2A and Video S2 with similar results, but the images shown in the figure panels and especially the video are still both so saturated that the quantification is difficult to interpret.) If there is evidence for wavefronts, it should be demonstrated explicitly by analysis of several clusters. For example, a figure of time-to-peak vs. position in the cluster demonstrating a propagating wave would satisfy this. Currently, I do not see any wavefronts in this data.

(2) The other wavefront claim relates to biofilms, and the relevant data is presented in Fig. S4 (and I believe also in what is now Video S8, but no supplemental video legends are provided, and this video is not cited in text.) As before, I cannot discern any wavefronts in the image and video provided; Reviewer 1 was also not able to detect wave propagation in this video by kymograph. Some mean squared displacements are shown in Fig. 7. As before, the methods for how these were obtained are not clearly documented either in this manuscript or in the BioRXiv preprint linked in the initial response to reviewers, and since wavefronts are not evident in the video it is hard to understand what is being measured here- radial distance from where? (The methods section mentions radial distance from the substrate, this should mean Z position above the imaging surface, and no wavefronts are evident in Z in the figure panels or movie.) Thus, clear demonstration of these wavefronts is still missing here as well.

Fourth, I have some specific questions about the study of blue light stress and the use of PI as a cell viability indicator:

(1) The logic of this paper includes the premise that blue light exposure is a stressor under the experimental conditions employed in the paper. Although it is of course generally true that blue light can be damaging to bacteria, this is dependent on light power and dosage. The control I recommended above, staining cells with PI in the presence and absence of blue light, will also allow the authors to confirm that this blue light treatment is indeed a stressor- the PI staining would be expected to increase in the presence of blue light if this is so.

(2) The presence of ThT may complicate the study of the blue light stress response, since ThT enhances the photodynamic effects of blue light in E. coli (Bondia et al. 2021 Chemical Communications). The authors could investigate ThT toxicity under these conditions by staining cells with PI after exposing them to blue light with or without ThT staining.

(3) In my initial review, I wrote the following: "In Figures 4D - E, the interpretation of this experiment can be confounded by the fact that PI uptake can sometimes be seen in bacterial cells with high membrane potential (Kirchhoff & Cypionka 2017 J Microbial Methods); the interpretation is that high membrane potential can lead to increased PI permeability. Because the membrane potential is largely higher throughout blue light treatment in the ∆kch mutant (Fig. 3[BC]), this complicates the interpretation of this experiment." In their response, the authors suggested that these results are not relevant in this case because "In our experiment methodology, cell death was not forced on the cells by introducing an extra burden or via anoxia." However, the logic of the paper is that the cells are in fact dying due to an imposed external stressor, which presumably also confers an increased burden as the cells try to deal with the stress. Instead, the authors should simply use a parallel method to confirm the results of PI staining. For example, the experiment could be repeated with other stains, or the viability of blue light-treated cells could be addressed more directly by outgrowth or colony-forming unit assays.

The CFU assay suggested above has the additional advantage that it can also be performed on planktonic cells in liquid culture that are exposed to blue light. If, as the paper suggests, a protective response to blue light is being coordinated at the biofilm level by these membrane potential fluctuations, the WT strain might be expected to lose its survival advantage vs. the ∆kch mutant in the absence of a biofilm.

Fifth, in several cases the data are presented in a way that are difficult to interpret, or the paper makes claims that are different to observe in the data:

(1) The authors suggest that the ThT and TMRM traces presented in Fig. S1D have similar shapes, but this is not obvious to me- the TMRM curve has very little decrease after the initial peak and only a modest, gradual rise thereafter. The authors suggest that this is due to increased TMRM photobleaching, but I would expect that photobleaching should exacerbate the signal decrease after the initial peak. Since this figure is used to support the use of ThT as a membrane potential indicator, and since this is the only alternative measurement of membrane potential presented in text, the authors should discuss this discrepancy in more detail.

(2) The comparison of single cells to microcolonies presented in figures 1B and D still needs revision:

First, both reviewer 1 and I commented in our initial reviews that the ThT traces, here and elsewhere, should not be normalized- this will help with the interpretation of some of the claims throughout the manuscript.

Second, the way these figures are shown with all traces overlaid at full opacity makes it very difficult to see what is being compared. Since the point of the comparison is the time to first peak (and the standard deviation thereof), histograms of the distributions of time to first peak in both cases should be plotted as a separate figure panel.<br /> Third, statistical significance tests ought to be used to evaluate the statistical strength of the comparisons between these curves. The authors compare both means and standard deviations of the time to first peak, and there are appropriate statistical tests for both types of comparisons.

(3) The authors claim that the curve shown in Fig. S4B is similar to the simulation result shown in Fig. 7B. I remain unconvinced that this is so, particularly with respect to the kinetics of the second peak- at least it seems to me that the differences should be acknowledged and discussed. In any case, the best thing to do would be to move Fig. S4B to the main text alongside Fig. 7B so that the readers can make the comparison more easily.

(4) As I wrote in my first review, in the discussion of voltage-gated calcium channels, the authors refer to "spiking events", but these are not obvious in Figure S3E. Although the fluorescence intensity changes over time, these fluctuations cannot be distinguished from measurement noise. A no-light control could help clarify this.

(5) In the lower irradiance conditions in Fig. 4A, the ThT dynamics are slower overall, and it looks like the ThT intensity is beginning to rise at the end of the measurement. The authors write that no second peak is observed below an irradiance threshold of 15.99 µW/mm2. However, could a more prominent second peak be observed in these cases if the measurement time was extended? Additionally, the end of these curves looks similar to the curve in Fig. S4B, in which the authors write that the slow rise is evidence of the presence of a second peak, in contrast to their interpretation here.

Additional considerations:

(1) The analysis and interpretation of the first peak, and particularly of the time-to-fire data is challenging throughout the manuscript the time resolution of the data set is quite limited. It seems that a large proportion of cells have already fired after a single acquisition frame. It would be ideal to increase the time resolution on this measurement to improve precision. This could be done by imaging more quickly, but that would perhaps necessitate more blue light exposure; an alternative is to do this experiment under lower blue light irradiance where the first spike time is increased (Figure 4A).

(2) The authors suggest in the manuscript that "E. coli biofilms use electrical signalling to coordinate long-range responses to light stress." In addition to the technical caveats discussed above, I am missing a discussion about what these responses might be. What constitutes a long-range response to light stress, and are there known examples of such responses in bacteria?

(3) The presence of long-range blue light responses can also be interrogated experimentally, for example, by repeating the Live/Dead experiment in planktonic culture or the single-cell condition. If the protection from blue light specifically emerges due to coordinated activity of the biofilm, the ∆kch mutant would not be expected to show a change in Live/Dead staining in non-biofilm conditions. The CFU experiment I mentioned above could also implicate coordinated long-range responses specifically, if biofilms and liquid culture experiments can be compared (although I know that recovering cells from biofilms is challenging.)

4. At the end of the results section, the authors suggest a critical biofilm size of only 4 μm for wavefront propagation (not much larger than a single cell!) The authors show responses for various biofilm sizes in Fig. 2C, but these are all substantially larger (and this figure also does not contain wavefront information.) Are there data for cell clusters above and below this size that could support this claim more directly?

(5) In Fig. 4C, the overall trajectories of extracellular potassium are indeed similar, but the kinetics of the second peak of potassium are different than those observed by ThT (it rises minutes earlier)- is this consistent with the idea that Kch is responsible for that peak? Additionally, the potassium dynamics also include the first ThT peak- is this surprising given that the Kch channel has no effect on this peak according to the model?

Detailed comments:

Why are Fig. 2A and Video S2 called a microcluster, whereas Video S3, which is smaller, is called a biofilm?

"We observed a spontaneous rapid rise in spikes within cells in the center of the biofilm" (Line 140): What does "spontaneous" mean here?

"This demonstrates that the ion-channel mediated membrane potential dynamics is a light stress relief process.", "E. coli cells employ ion-channel mediated dynamics to manage ROS-induced stress linked to light irradiation." (Line 268 and the second sentence of the Fig. 4F legend): This claim is not well-supported. There are several possible interpretations of the catalase experiment (which should be discussed); this experiment perhaps suggests that ROS impacts membrane potential but does not indicate that these membrane potential fluctuations help the cells respond to blue light stress. The loss of viability in the ∆kch mutant might indicate a link between these membrane potential experiments and viability, but it is hard to interpret without the no light controls I mention above.

"The model also predicts... the external light stress" (Lines 338-341): Please clarify this section. Where does this prediction arise from in the modeling work? Second, I am not sure what is meant by "modulates the light stress" or "keeps the cell dynamics robust to the intensity of external light stress" (especially since the dynamics clearly vary with irradiance, as seen in Figure 4A).

"We hypothesized that E. coli not only modulates the light-induced stress but also handles the increase of the ROS by adjusting the profile of the membrane potential dynamics" (Line 347): I am not sure what "handles the ROS by adjusting the profile of the membrane potential dynamics" means. What is meant by "handling" ROS? Is the hypothesis that membrane potential dynamics themselves are protective against ROS, or that they induce a ROS-protective response downstream, or something else? Later the authors write that changes in the response to ROS in the model agree with the hypothesis, but just showing that ROS impacts the membrane potential does not seem to demonstrate that this has a protective effect against ROS.

"Mechanosensitive ion channels (MS) are vital for the first hyperpolarization event in E. coli." (Line 391): This is misleading- mechanosensitive ion channels totally ablate membrane potential dynamics, they don't have a specific effect on the first hyperpolarization event. The claim that mechanonsensitive ion channels are specifically involved in the first event also appears in the abstract.

Also, the apparent membrane potential is much lower even at the start of the experiment in these mutants (Fig. 6C-D)- is this expected? This seems to imply that these ion channels also have a blue light-independent effect.

Throughout the paper, there are claims that the initial ThT spike is involved in "registering the presence of the light stress" and similar. What is the evidence for this claim?

"We have presented much better quantitative agreement of our model with the propagating wavefronts in E. coli biofilms..." (Line 619): It is not evident to me that the agreement between model and prediction is "much better" in this work than in the cited work (reference 57, Hennes et al. 2023). The model in Figure 4 of ref. 57 seems to capture the key features of their data.

In methods, "Only cells that are hyperpolarized were counted in the experiment as live" (Line 745): what percentage of cells did not hyperpolarize in these experiments?

Some indication of standard deviation (error bars or shading) should be added to all figures where mean traces are plotted.

Video S8 is very confusing- why does the video play first forwards and then backwards? It is easy to misinterpret this as a rise in the intensity at the end of the experiment.

This sentence shows a deep theme for Othello and for Lago's character as a whole, the idea of jealousy. The way Lago describes Cassio is that of someone lesser than himself and maybe even lesser a person. Attacking his origin, a "Florentine" as some form of insult, calling Cassio not one of us. Also he attacks Cassios Battle knowledge saying he's "Never set a squadron in the field Nor the division of a battle knows". These remarks show Lagos jealousy as he try's to tear down Cassio to make himself feel better and look more suitable a lieutenant.

eLife Assessment

This is a fundamental study that provides a detailed single-cell transcriptomic and epigenomic map of the mouse trabecular meshwor, identifying three distinct trabecular meshwor subtypes with specific functional roles. It links the glaucoma-associated transcription factor LMX1B to mitochondrial regulation in TM3 cells and demonstrates that nicotinamide treatment prevents IOP elevation in Lmx1bV265D/+ mutant mice, highlighting a potential metabolic therapeutic strategy for glaucoma. This convincing work would be further supported by data that link the transcriptional data with mitochondrial functional assays.

Reviewer #1 (Public review):

Summary:

This study provides a comprehensive single-cell and multiomic characterization of trabecular meshwork (TM) cells in the mouse eye, a structure critical to intraocular pressure (IOP) regulation and glaucoma pathogenesis. Using scRNA-seq, snATAC-seq, immunofluorescence, and in situ hybridization, the authors identify three transcriptionally and spatially distinct TM cell subtypes. The study further demonstrates that mitochondrial dysfunction, specifically in one subtype (TM3), contributes to elevated IOP in a genetic mouse model of glaucoma carrying a mutation in the transcription factor Lmx1b. Importantly, treatment with nicotinamide (vitamin B3), known to support mitochondrial health, prevents IOP elevation in this model. The authors also link their findings to human datasets, suggesting the existence of analogous TM3-like cells with potential relevance to human glaucoma.

Strengths:

The study is methodologically rigorous, integrating single-cell transcriptomic and chromatin accessibility profiling with spatial validation and in vivo functional testing. The identification of TM subtypes is consistent across mouse strains and institutions, providing robust evidence of conserved TM cell heterogeneity. The use of a glaucoma model to show subtype-specific vulnerability, combined with a therapeutic intervention-gives the study strong mechanistic and translational significance. The inclusion of chromatin accessibility data adds further depth by implicating active transcription factors such as LMX1B, a gene known to be associated with glaucoma risk. The integration with human single-cell datasets enhances the potential relevance of the findings to human disease.

Weaknesses:

Although the LMX1B transcription factor is implicated as a key regulator in TM3 cells, its role in directly controlling mitochondrial gene expression is not fully explored. Additional analysis of motif accessibility or binding enrichment near relevant target genes could substantiate this mechanistic link. The therapeutic effect of vitamin B3 is clearly demonstrated phenotypically, but the underlying cellular and molecular mechanisms remain somewhat underdeveloped - for instance, changes in mitochondrial function, oxidative stress markers, or NAD+ levels are not directly measured. While the human relevance of TM3 cells is suggested through marker overlap, more quantitative approaches, such as cell identity mapping or gene signature scoring in human datasets, would strengthen the translational connection.

Overall, this is a compelling and carefully executed study that offers significant advances in our understanding of TM cell biology and its role in glaucoma. The integration of multimodal data, disease modeling, and therapeutic testing represents a valuable contribution to the field. With additional mechanistic depth, the study has the potential to become a foundational resource for future research into IOP regulation and glaucoma treatment.

Reviewer #2 (Public review):

Summary:

This elegant study by Tolman and colleagues provides fundamental findings that substantially advance our knowledge of the major cell types within the limbus of the mouse eye, focusing on the aqueous humor outflow pathway. The authors used single-cell and single-nuclei RNAseq to very clearly identify 3 subtypes of the trabecular meshwork (TM) cells in the mouse eye, with each subtype having unique markers and proposed functions. The U. Columbia results are strengthened by an independent replication in a different mouse strain at a separate laboratory (Duke). Bioinformatics analyses of these expression data were used to identify cellular compartments, molecular functions, and biological processes. Although there were some common pathways among the 3 subtypes of TM cells (e.g., ECM metabolism), there also were distinct functions. For example:

• TM1 cell expression supports heavy engagement in ECM metabolism and structure, as well as TGFβ2 signaling.

• TM2 cells were enriched in laminin and pathways involved in phagocytosis, lysosomal function, and antigen expression, as well as End3/VEGF/angiopoietin signaling.

• TM3 cells were enriched in actin binding and mitochondrial metabolism.

They used high-resolution immunostaining and in situ hybridization to show that these 3 TM subtypes express distinct markers and occupy distinct locations within the TM tissue. The authors compared their expression data with other published scRNAseq studies of the mouse as well as the human aqueous outflow pathway. They used ATAC-seq to map open chromatin regions in order to predict transcription factor binding sites. Their results were also evaluated in the context of human IOP and glaucoma risk alleles from published GWAS data, with interesting and meaningful correlations. Although not discussed in their manuscript, their expression data support other signaling pathways/ proteins/ genes that have been implicated in glaucoma, including: TGFβ2, BMP signaling (including involvement of ID proteins), MYOC, actin cytoskeleton (CLANs), WNT signaling, etc.

In addition to these very impressive data, the authors used scRNAseq to examine changes in TM cell gene expression in the mouse glaucoma model of mutant Lmxb1-induced ocular hypertension. In man, LMX1B is associated with Nail-Patella syndrome, which can include the development of glaucoma, demonstrating the clinical relevance of this mouse model. Among the gene expression changes detected, TM3 cells had altered expression of genes associated with mitochondrial metabolism. The authors used their previous experience using nicotinamide to metabolically protect DBA2/J mice from glaucomatous damage, and they hypothesized that nicotinamide supplementation of mutant Lmx1b mice would help restore normal mitochondrial metabolism in the TM and prevent Lmx1b-mediated ocular hypertension. Adding nicotinamide to the drinking water significantly prevented Lmxb1 mutant mice from developing high intraocular pressure. This is a laudable example of dissecting the molecular pathogenic mechanisms responsible for a disease (glaucoma) and then discovering and testing a potential therapy that directly intervenes in the disease process and thereby protects from the disease.

Strengths:<br /> There are numerous strengths in this comprehensive study including:<br /> • Deep scRNA sequencing that was confirmed by an independent dataset in another mouse strain at another university.<br /> • Identification and validation of molecular markers for each mouse TM cell subset along with localization of these subsets within the mouse aqueous outflow pathway.<br /> • Rigorous bioinformatics analysis of these data as well as comparison of the current data with previously published mouse and human scRNAseq data.<br /> • Correlating their current data with GWAS glaucoma and IOP "hits".<br /> • Discovering gene expression changes in the 3 TM subgroups in the mouse mutant Lmx1b model of glaucoma.<br /> • Further pursuing the indication of dysfunctional mitochondrial metabolism in TM3 cells from Lmx1b mutant mice to test the efficacy of dietary supplementation with nicotinamide. The authors nicely demonstrate the disease modifying efficacy of nicotinamide in preventing IOP elevation in these Lmx1b mutant mice, preventing the development of glaucoma. These results have clinical implications for new glaucoma therapies.

Weaknesses:<br /> • Occasional over-interpretation of data. The authors have used changes in gene expression (RNAseq) to implicate functions and signaling pathways. For example: they have not directly measured "changes in metabolism", "mitochondrial dysfunction" or "activity of Lmx1b".<br /> • In their very thorough data set, there is enrichment of or changes in gene expression that support other pathways that have been previously reported to be associated with glaucoma (such as TGFβ2, BMP signaling, actin cytoskeletal organization (CLANs), WNT signaling, ossification, etc. that appears to be a lost opportunity to further enhance the significance of this work.

Reviewer #3 (Public review):

Summary:In this study, the authors perform multimodal single-cell transcriptomic and epigenomic profiling of 9,394 mouse TM cells, identifying three transcriptionally distinct TM subtypes with validated molecular signatures. TM1 cells are enriched for extracellular matrix genes, TM2 for secreted ligands supporting Schlemm's canal, and TM3 for contractile and mitochondrial/metabolic functions. The transcription factor LMX1B, previously linked to glaucoma, shows the highest expression in TM3 cells and appears to regulate mitochondrial pathways. In Lmx1bV265D mutant mice, TM3 cells exhibit transcriptional signs of mitochondrial dysfunction associated with elevated IOP. Notably, vitamin B3 treatment significantly mitigates IOP elevation, suggesting a potential therapeutic avenue.

This is an excellent and collaborative study involving investigators from two institutions, offering the most detailed single-cell transcriptomic and epigenetic profiling of the mouse limbal tissues-including both TM and Schlemm's canal (SC), from wild-type and Lmx1bV265D mutant mice. The study defines three TM subtypes and characterizes their distinct molecular signatures, associated pathways, and transcriptional regulators. The authors also compare their dataset with previously published murine and human studies, including those by Van Zyl et al., providing valuable cross-species insights.

Strengths:

(1) Comprehensive dataset with high single-cell resolution<br /> (2) Use of multiple bioinformatic and cross-comparative approaches<br /> (3) Integration of 3D imaging of TM and SC for anatomical context<br /> (4) Convincing identification and validation of three TM subtypes using molecular markers.

Weaknesses:

(1) Insufficient evidence linking mitochondrial dysfunction to TM3 cells in Lmx1bV265D mice: While the identification of TM3 cells as metabolically specialized and Lmx1b-enriched is compelling, the proposed link between Lmx1b mutation and mitochondrial dysfunction remains underdeveloped. It is unclear whether mitochondrial defects are a primary consequence of Lmx1b-mediated transcriptional dysregulation or a secondary response to elevated IOP. Additional evidence is needed to clarify whether Lmx1b directly regulates mitochondrial genes (e.g., via ChIP-seq, motif analysis, or ATAC-seq), or whether mitochondrial changes are downstream effects.<br /> Furthermore, the protective effects of nicotinamide (NAM) are interpreted as evidence of mitochondrial involvement, but no direct mitochondrial measurements (e.g., immunostaining, electron microscopy, OCR assays) are provided. It is essential to validate mitochondrial dysfunction in TM3 cells using in vivo functional assays to support the central conclusion of the paper. Without this, the claim that mitochondrial dysfunction drives IOP elevation in Lmx1bV265D mice remains speculative. Alternatively, authors should consider revising their claims that mitochondrial dysfunction in these mice is a central driver of TM dysfunction.

(2) Mechanism of NAM-mediated protection is unclear: The manuscript states that NAM treatment prevents IOP elevation in Lmx1bV265D mice via metabolic support, yet no data are shown to confirm that NAM specifically rescues mitochondrial function. Do NAM-treated TM3 cells show improved mitochondrial integrity? Are reactive oxygen species (ROS) reduced? Does NAM also protect RGCs from glaucomatous damage? Addressing these points would clarify whether the therapeutic effects of NAM are indeed mitochondrial.

(3) Lack of direct evidence that LMX1B regulates mitochondrial genes: While transcriptomic and motif accessibility analyses suggest that LMX1B is enriched in TM3 cells and may influence mitochondrial function, no mechanistic data are provided to demonstrate direct regulation of mitochondrial genes. Including ChIP-seq data, motif enrichment at mitochondrial gene loci, or perturbation studies (e.g., Lmx1b knockout or overexpression in TM3 cells) would greatly strengthen this central claim.

(4)Focus on LMX1B in Fig. 5F lacks broader context: Figure 5F shows that several transcription factors (TFs)-including Tcf21, Foxs1, Arid3b, Myc, Gli2, Patz1, Plag1, Npas2, Nr1h4, and Nfatc2-exhibit stronger positive correlations or motif accessibility changes than LMX1B. Yet the manuscript focuses almost exclusively on LMX1B. The rationale for this focus should be clarified, especially given LMX1B's relatively lower ranking in the correlation analysis. Were the functions of these other highly ranked TFs examined or considered in the context of TM biology or glaucoma? Discussing their potential roles would enhance the interpretation of the transcriptional regulatory landscape and demonstrate the broader relevance of the findings.

Other weaknesses:

(1) In abstract, they say a number of 9,394 wild-type TM cell transcriptomes. The number of Lmx1bV265D/+ TM cell transcriptomes analyzed is not provided. This information is essential for evaluating the comparative analysis and should be clearly stated in the Abstract and again in the main text (e.g., lines 121-123). Including both wild-type and mutant cell counts will help readers assess the balance and robustness of the dataset.

(2) Did the authors monitor mouse weight or other health parameters to assess potential systemic effects of treatment? It is known that the taste of compounds in drinking water can alter fluid or food intake, which may influence general health. Also, does Lmx1bV265D/+ have mice exhibit non-ocular phenotypes, and if so, does nicotinamide confer protection in those tissues as well? Additionally, starting the dose of the nicotinamide at postnatal day 2, how long the mice were treated with water containing nicotinamide, and after how many days or weeks IOP was reduced, and how long the decrease in the IOP was sustained.<br /> (3) While the IOP reduction observed in NAM-treated Lmx1bV265D/+ mice appears statistically significant, it is unclear whether this reflects meaningful biological protection. Several untreated mice exhibit very high IOP values, which may skew the analysis. The authors should report the mean values for IOP in both untreated and NAM-treated groups to clarify the magnitude and variability of the response.<br /> (4) Additionally, since NAM has been shown to protect RGCs in other glaucoma models directly, the authors should assess whether RGCs are preserved in NAM-treated Lmx1b V265D/+ mice. Demonstrating RGC protection would support a synergistic effect of NAM through both IOP reduction and direct neuroprotection, strengthening the translational relevance of the treatment.<br /> (5) Can the authors add any other functional validation studies to explore to understand the pathways enriched in all the subtypes of TM1, TM2, and TM3 cells, in addition to the ICH/IF/RNAscope validation?<br /> (6) The authors should include a representative image of the limbal dissection. While Figure S1 provides a schematic, mouse eyes are very small, and dissecting unfixed limbal tissue is technically challenging. It is also difficult to reconcile the claim that the majority of cells in the limbal region are TM and endothelium. As shown in Figure S6, DAPI staining suggests a much higher abundance of scleral cells compared to TM cells within the limbal strip. Additional clarification or visual evidence would help validate the dissection strategy and cellular composition of the captured region.

eLife Assessment

This is a valuable methodological contribution towards accurate characterization of viral genetic diversity using long-read sequencing and unique molecular identifiers (UMIs). However, the methods are currently incomplete and the sensitivity is not rigorously demonstrated. Addressing these gaps would strengthen the manuscript and make it a key addition to the field.

Reviewer #1 (Public review):

Tamao et al. aimed to quantify the diversity and mutation rate of the influenza (PR8 strain) in order to establish a high-resolution method for studying intra-host viral evolution. To achieve this, the authors combined RNA sequencing with single-molecule unique molecular identifiers (UMIs) to minimize errors introduced during technical processing. They proposed an in vitro infection model with a single viral particle to represent biological genetic diversity, alongside a control model using in vitro transcribed RNA for two viral genes, PB2 and HA.

Through this approach, the authors demonstrated that UMIs reduced technical errors by approximately tenfold. By analyzing four viral populations and comparing them to in vitro transcribed RNA controls, they estimated that ~98.1% of observed mutations originated from viral replication rather than technical artifacts. Their results further showed that most mutations were synonymous and introduced randomly. However, the distribution of mutations suggested selective pressures that favored certain variants. Additionally, comparison with a closely related influenza strain (A/Alaska/1935) revealed two positively selected mutations, though these were absent in the strain responsible for the most recent pandemic (CA01).

Overall, the study is well-designed, and the interpretations are strongly supported by the data. However, the following clarifications are recommended:

(1) The methods section is overly brief. Even if techniques are cited, more experimental details should be included. For example, since the study focuses heavily on methodology, details such as the number of PCR cycles in RT-PCR or the rationale for choosing HA and PB2 as representative in vitro transcripts should be provided.

(2) Information on library preparation and sequencing metrics should be included. For example, the total number of reads, any filtering steps, and quality score distributions/cutoff for the analyzed reads.

(3) In the Results section (line 115, "Quantification of error rate caused by RT"), the mutation rate attributed to viral replication is calculated. However, in line 138, it is unclear whether the reported value reflects PB2, HA, or both, and whether the comparison is based on the error rate of the same viral RNA or the mean of multiple values (as shown in Figure 3A). Please clarify whether this number applies universally to all influenza RNAs or provide the observed range.

(4) Since the T7 polymerase introduced errors are only applied to the in vitro transcription control, how were these accounted for when comparing mutation rates between transcribed RNA and cell-culture-derived virus?

(5) Figure 2 shows that a UMI group size of 4 has an error rate of zero, but this group size is not mentioned in the text. Please clarify.

Reviewer #2 (Public review):

Summary:

This manuscript presents a technically oriented application of UMI-based long-read sequencing to study intra-host diversity in influenza virus populations. The authors aim to minimize sequencing artifacts and improve the detection of rare variants, proposing that this approach may inform predictive models of viral evolution. While the methodology appears robust and successfully reduces sequencing error rates, key experimental and analytical details are missing, and the biological insight is modest. The study includes only four samples, with no independent biological replicates or controls, which limits the generalizability of the findings. Claims related to rare variant detection and evolutionary selection are not fully supported by the data presented.

Strengths:

The study addresses an important technical challenge in viral genomics by implementing a UMI-based long-read sequencing approach to reduce amplification and sequencing errors. The methodological focus is well presented, and the work contributes to improving the resolution of low-frequency variant detection in complex viral populations.

Weaknesses:

The application of UMI-based error correction to viral population sequencing has been established in previous studies (e.g., in HIV), and this manuscript does not introduce a substantial methodological or conceptual advance beyond its use in the context of influenza.

The study lacks independent biological replicates or additional viral systems that would strengthen the generalizability of the conclusions. Potential sources of technical error are not explored or explicitly controlled. Key methodological details are missing, including the number of PCR cycles, the input number of molecules, and UMI family size distributions. These are essential to support the claimed sensitivity of the method.

The assertion that variants at {greater than or equal to}0.1% frequency can be reliably detected is based on total read count rather than the number of unique input molecules. Without information on UMI diversity and family sizes, the detection limit cannot be reliably assessed.

Although genetic variation is described, the functional relevance of observed mutations in HA and NA is not addressed or discussed in the context of known antigenic or evolutionary features of influenza. The manuscript is largely focused on technical performance, with limited exploration of the biological implications or mechanistic insights into influenza virus evolution.

The experimental scale is small, with only four viral populations derived from single particles analyzed. This limited sample size restricts the ability to draw broader conclusions about quasispecies dynamics or evolutionary pressures.

This saved lots of time when I was programming in Java and Python. The loop can save lots of time, and programmers do not need to repeat for so many times. We can even use the for loop for counting numbers and print something with patterns.

The author is giving a sense of how the dynamic works in the band to the audience who is reading.

They are showing frustration of the disrespect they get from player's on the football team. All they honestly won't is some recognition and respect for what they work so hard for.

This can be a relation for many and it could be a connection to anything. The thrill and excitement you get from doing something so fun also come's with it's cons of being terrified of what may happen.

This quote is giving a sense of frustration of the town that she is apart of. And also claiming that people aren't given a lot of opportunity which gives a sense of how little the town may be.

Being a cheerleader, I understand these words completely. When I'm able to show off skills my team and I have been working on for long periods of time, I always feel accomplished and proud.

I can relate to going through tough times with teammates. These are the times that you bond the most over your shared interest/ passion because everyone supports each other through really rough patches.

Considering how small the school is, the handful of people who share a common interest are bound to bond over it. Programs at larger schools could become more disconnected.

This highlights conflicts and aspirations. The sentence "hungry for opportunities beyond his hometown" suggest ambition and a desire to explore beyond what he is used to.

Sometimes it can be hard to understand different peoples experiences when it comes to things like anxiety. However when you know you are surrounded by people who understand and care for you, it is easier to get comfortable and begin to feel more at home.

It seems like Ashlee enjoys surrounding herself with people of common interest in order to push each other towards a common goal. It always feels good to be surrounded by people who have the same if not more drive than you do.

This is something I find hard to relate to. I'm originally from Burbank CA where walt Disney studios and universal studios are right next to each other so everything is typically crowded and loud.

Julia says this when describing how the band room feels like a safe space to her.

Not everyone is a family in band, some others just play music just to play music.

Recruiting new members for band is something that Julia and Nina want to expand on to show how this family thhing can be very useful for others.

The person saying this shows that theyre closest friends are from band. Almost like a family.

Julia feels more comfortable by meeting new people, getting our her shell.

Here Julia is explaining how band gives her a support system even though it can be stressful.

Julia is talking about her experience with the high school band and how nervous she felt.

it is great having so much to access with the internet you can look up whatever your searching and you can even use ai to find something very specific it is hard to find just by searching in the search bar

Such a disparity is not a matter of whether there is love or not, but rather a practical constraint on whether financial circumstances allow parents to devote time and energy. I can appreciate the importance of family support for academic performance, and parents from low-income families are not unwilling to provide that support — instead, they have been drained of their capacity by the pressure of making ends meet.

During group projects, I noticed that classmates from highly educated families tend to excel at expressing their views clearly and grasping academic concepts quickly. Only now do I realize this is the result of long-term immersion in a language-rich environment. Such early language advantages translate into subsequent reading skills, writing abilities, and even critical thinking. In contrast, children from low-income families, due to a lack of such linguistic input, gradually fall behind academically — and this gap is far harder to bridge than the one addressed by taking after-school classes.

I came into contact with children from impoverished communities during my volunteer work. Some of them did have issues with inattention and emotional sensitivity. At the time, I thought these were personality issues, but now I realize they are physical and psychological trauma caused by an impoverished environment. Addressing academic gaps cannot rely solely on after-school tutoring provided by schools; it more importantly requires society to tackle basic issues faced by low-income families, such as nutrition, housing safety.

Such spending disparities are not a choice between luxury and frugality, but rather a gap in whether one can meet developmental needs. During my study-abroad experience, classmates from affluent families around me often gained access to resources like research internships and overseas exchange programs much earlier. In contrast, financially constrained classmates might have to work multiple jobs at the same time to support their studies, making it hard for them to devote time to enhancing their soft skills. Economic resources thus exert an invisible monopoly over individuals' development opportunities.

This data shocked me profoundly — when children first enter the formal education system, the income-related gaps in early literacy skills actually far exceed racial gaps, and are equivalent to an average child’s entire year of kindergarten learning outcomes. I have observed the "differences in starting points" among students from different countries, but never realized that the impact of "family income" on educational starting points could be so direct and profound. This shows that the pursuit of "educational equity" does not start from primary or secondary school; instead, it is already shaped in the family environment during children’s preschool years. Children from low-income families, right from the "starting line," face insurmountable barriers.

Here are the 12 Principles of the Agile Manifesto, briefly stated:

Customer satisfaction – Deliver valuable software early and continuously.

Welcome change – Even late in development, embrace changing requirements.

Frequent delivery – Deliver working software regularly, from weeks to months.

Collaboration – Business people and developers must work together daily.

Motivated individuals – Build projects around trusted, supported team members.

Face-to-face communication – The most effective way to convey information.

Working software – The primary measure of progress.

Sustainable pace – Maintain a steady, continuous development rhythm.

Technical excellence – Continuous attention to quality improves agility.

Simplicity – Maximize the amount of work not done; keep things simple.

Self-organizing teams – The best designs and solutions emerge from them.

Regular reflection – Teams should reflect and adjust to improve effectiveness.

This helps us see the bigger picture and bigger impacts of collected data. The data isn't just one individual point that connects to a report, it is far more complex than that

I like the way they worded this. It connects databases to the past and present which helps when thinking about why or what the data represents and will be used for in the future.

I think this quote will inspire many people to follow their dreams no matter what. To never give up and hard work will pay off.

I think the fact that he never gave up and did what he loved no matter what the outcome was at the time, is very inspirational.

I think its amazing that he was so scared to take such a big risk and it all worked out for him in the end.

I thought this was interesting because for a while I wasn't sure what I wanted to do and felt hopeless with college.

I think that it's amazing how he dealt with such negative things before he was even born and now is such a big success is very encouraging.

this shows how these instances are of gender inequality can be so individually isolating, where you don't even realize the bigger picture and gender gap.

in this document

a person who makes and repairs things in iron by hand

bars/ tavern (places with beer)

The person as a "fugitive thing."

Makes me look to the often "invisible" work of women. It gestures toward the kind of intellectual labor that often goes unrecognized, especially when done by women. It reflects how Harsh’s deep archival and curatorial work, though not always expressed in traditional scholarly formats, was essential to shaping Black historical memory. The line reminds me of how much invisible labor women have done, collecting, preserving, mentoring, organizing knowledge, without being credited as authors or theorists.

Scattered things (people).

Every single image within this book has struck me. I think Helton has done an amazing job choosing what should be included image wise. Each one leaves me wanting more. The visual selections don’t just complement the text, they carry their own emotional and historical weight.

People as scattered things.

This challenges the intellectual (?) idea to center the famous. Instead we center the ORDINARY READER. To me this sentence reclaims the everyday intellectual lives of ordinary library patrons, people who can be eaisly overlooked. It's a quiet but important act of historical recovery.

I love this photo. It looks like a photo from a movie. I think it highlights the ordinary (the group of children by the counter, the librarians behind the counter, no one looking at the camera). This chapter really highlights the Ordinary Reader and their key importance.

I love the sentence "rage born of history's denial." I think it really captures the powerful, emotional force behind the intellectual resistance that collecting and archiving became. This isn't just academic curiosity held by collectors, it is knowledge-making that is driven by historical erasure and systematic injustice.

the glorification of these 3 men in a air battle that claimed the lives of 1563 men.

Through the inclusion of Elizabeth, in conjunction with the decaying description of Thames River, Eliot continues his complex commentary on love in modern society. First, Eliot begins this section of The Fire Sermon with the following lines: "The river sweats Oil and tar" (266-67). The contrast between the verb "to sweat"--a natural process to regulate temperature--and artificial/manmade substances such as "oil" or "tar" highlight the contradictory nature of River Thames. The once pure and sacred area now lies bare: the "nymphs have departed," the bank has lost its sole, and the water runs polluted. Now, Eliot's reference to Queen Elizabeth here ties to similar themes. For instance, she was once celebrated for her virginity. Society at the time was transfixed by her feminine purity, therefore hitting similar notes to the previously discussed trends of the land/river. However, her key connection to River Thames comes from Froude's Queen Elizabeth in her interaction with Leicester at the "water-party", seen through the eyes of De Quadra. He states, "In the afternoon, we were in a barge, watching the games on the river. She was alone with the Lord Robert and myself, on the poop, when they began to talk nonsense, and went so far, that Lorb Robert at l said, as I was on the spot there was no reason why they should not be married, if the queen pleased." Here, Elizabeth and Leicester are openly flirting, even suggesting marriage, with the river as the backdrop. This moment highlights a dramatic social scandal of the time given Leicester's past wife and complex relationship with Elizabeth. Here, it is being put on full display to the public eye. Therefore, Eliot continues to explore complex nature of love (as seen previously with the clerk and the typist) with this citation. Furthermore, as previously discussed, river Thames comes to mimic this decay alongside the thorny state of human relationships at the time through the unnatural pollution imagery.

It’s honestly really sad to see that women are often victims of homicide or violence, especially when it’s by their own family members. The fact alone is heartbreaking and shows how serious and personal this problem is. It makes me wonder if the government or police are doing anything to address and prevent this kind of violence.

I agree with this because debt is a big financial problem for many college students. Some students take out a lot of loans to pay for school, but later realize they can’t borrow any more money and end up dropping out.

This passage is eye opening because it shows how hard it is for some kids just to get to school. In the mountains of Peru, they have to walk for hours, and some can’t go at all because their families need them to work. It made me realize that going to school isn’t easy or possible for everyone. It also makes me wonder will they ever get buses? The schools know most of these families are poor, so why isn’t more being done to help them?

This brought me back to my Comm Strat class where we had to practice interviews with AI and then it would give us feedback. I actually really dislike doing this because a lot of what I said, the AI would mishear me even if I had really clear pronunciation. I think this article overall really shows how much the workforce is transforming and what I should be preparing for in the future.

Is a highly routinized services a poor fit with the needs and expectations of a diverse employee population.

Sometimes this can have the reverse of the effect that it is trying to have such as tokenism.

Personal vs. the organizations

Is there a regular IT staff or is it just the company?

This cartoon is showing that the public never learns from their past economic and financial mistakes. This illustrates the comparison of the Florida land boom, that ended in complete failure. Showing people’s desire to get rich faster, making them to fall into the same traps that lead to this type of mistakes.

This example of a change in a social segment is a great demonstration of a macro trend. The growing preference for salsa over ketchup represents demographic shifts. Another example of a macro trend is the rise in vegan and vegetarian foods, reflecting long-term changes in how we view health and the environment.

I wasn't sure how I was feeling about cold calling at first, however after observing the freshman English teacher today I think that she changed my mind about it. With my CT we don't meed to utilize cold calling all that often, but she must need to do it a lot because she was doing it masterfully and it made sure that many students were able to be involved in the discussion and that everyone understood.

This is a downfall because this does not require the students to really expand their thinking. Giving short answers and moving on means they can just word vomit a response and then move on. There is no additional thinking required to help them remember what's being discussed or open their minds more.

Questioning is not something I've thought of as a skill before now honestly, but it is just that. As I've been observing in my CT's classroom and some of the other English teachers' classes I've seen how they question their classes purposefully and masterfully, enabling them to elaborate on a topic and enlighten them further. When I am the one teaching though, I do not quite go as long as they do because I don't have the same abilities when it comes to questioning as they do.

fearmongering,

Using this to justify the rapid change in policy.

So confused on this one.

He fears that larger shipping companies based on the east coast could begin to operate on the west coast, crushing regional/local competition

why this specific representative? did he sponsor it? did he lobby it?

Telling people not to be scared. This therefore implies he is protecting the people from something.

Confusing wording I think, the shipping board denies that this act will drive foreign ships from our ports. However, isn't that the whole point of this law?

Then why put this law in place? why not allocate money and provide subsidies to produce vessels in the united states and then give them favorable treatment? Was this considered? Were there American ships to take their place at this time?

• Commercial business men
• American Citizens who fear rising prices
• Shipping companies who reject higher maintenance costs
• The government wanting to contract out shipping to other nations for cheaper prices.

AMERICAN EXCEPTIONALISM...

Making an enemy of foreign shipping lines, using this as the basis to exclude them from continental US shipping.

Fear mongering, Extensive fear mongering.

More interesting rhetoric.

American exceptionalism is on full display here. Interesting rhetoric to use regarding a global industry.

People can refer to a multitude of things. - The government themselves necessitating a wartime fleet to support the US military - American shipping companies interested in expanding profits for themselves. - American shipping unions lobbying to defend American workers and jobs. - I can't really figure out why the American populous would want this.

Another way to show the controversiality of the law.

Potentially hard to determine what the 'tangible' injury is. Refused to listen to dissenting arguments, rejecting 'opinions of reason'.

Senator Jones comes across as arrogant in stating that they came there not to rebuke his argument, but to blow of their steam.

The author of the legislation, is said he used this to get re-elected to congress.

the jones act

opinion of the dissenters, aka trade business men who use waterways to ship goods.

sounds awfully like (vertical) integration in engineering practices

engaging all learners is the most essential thing! If a group of students does not feel like they are getting it or aren't feeling into the activity they won't try and they may not grasp the lesson. If we make sure to engage all students and make sure they stay attentive it will make things so much easier on everyone in the future!

I like this wordage because adapting and manipulating certain aspects of curriculums is how we make sure that all our students are getting what we are teaching. I feel that this should be standard honestly, because no two students are going to learn exactly the same.

Reminds me of Romeo and Juliet, the way it reminds me of Romeo and Juliet is in the second paragraph when the author says "she or he attaches strong feelings to the perfectly wonderful image they have created". Which compared to the story of Romeo and Juliet, in this case it would be Romeo catching feelings for Juliet. I don't have any personal experiences. I have learned that sometimes you may think you are in love but it is kinda a hallucination.

The part of this statement that stuck out to me was the emphasis on decision making. I wonder why the author brings this up so often. What about scientific thinking leads to better decision making?